Heterodimeric immunoglobulins

ABSTRACT

The present application is directed to heterodimeric antibodies and methods of use.

INCORPORATION BY REFERENCE

The following applications are hereby incorporated by reference in theirentirety: U.S. patent application Ser. No. 11/410,540, filed Apr. 25,2006, which claims priority to U.S. Provisional Patent Application No.60/792,645, filed Apr. 17, 2006, U.S. Provisional Patent Application No.60/782,244, filed Mar. 13, 2006, U.S. Provisional Patent Application No.60/776,847, filed Feb. 24, 2006, and U.S. Provisional Patent ApplicationNo. 60/677,583, filed May 3, 2005; and U.S. patent application Ser. No.11/411,003 (issued as U.S. Pat. No. 7,592,429), filed Apr. 25, 2006,which claims priority to U.S. Provisional Patent Application No.60/792,645, filed Apr. 17, 2006, U.S. Provisional Patent Application No.60/782,244, filed Mar. 13, 2006, U.S. Provisional Patent Application No.60/776,847, filed Feb. 24, 2006, and U.S. Provisional Patent ApplicationNo. 60/677,583, filed May 3, 2005. The following applications also arehereby incorporated by reference: U.S. patent application Ser. No.12/212,327, filed Sep. 17, 2008, which claims priority to U.S.Provisional Patent Application No. 60/973,024, filed Sep. 17, 2007; andU.S. patent application Ser. No. 12/811,171, filed Jun. 29, 2010, whichis a U.S. National Phase Application pursuant to 35 U.S.C. §371 ofInternational Patent Application No. PCT/US08/86864, filed on Dec. 15,2008, which claims priority to U.S. Provisional Patent Application No.61/013,917, filed Dec. 14, 2007.

TECHNICAL FIELD OF THE INVENTION

The invention generally relates to methods of making and usingheterodimeric antibodies for the treatment of disorders associated withlow bone mineral density.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: ASCII (text) file named“47233A1_SubSeqListing.txt,” 919,811 bytes, created on Feb. 6, 2014.

BACKGROUND OF THE INVENTION

The development of bispecific antibodies as therapeutic agents for humandiseases has great clinical potential. Bispecific antibodies cansimultaneously recognize two different antigens, neutralize differentpathogenic mediators, recruit different type of effector cells, andmodulate signal pathways. However, production of bispecific antibodieshas been very challenging. The broad application of bispecificantibodies has been hindered by the difficulties of developing aplatform for producing bispecific antibodies that exhibit favorablehalf-life, high stability, lack of immunogenicity, and feasibilities forlarge scale manufacturing and purification. Promising bispecificantibodies formats such as DVD-Ig (Dual Variable Domain Ig) (NatureBiotechnology 25, 1290-1297 (2007)); Cross-over Ig [Schaefer W et al(2011) PNAS 108(27): 11187-11192]; Two-in-One Ig (Science 2009, 323,1610); BiTE® antibodies [PNAS 92(15):7021-7025; 1995] allow theproduction of a bispecific antibody, but they do have different kinds ofliabilities.

SUMMARY OF THE INVENTION

Described herein are methods of generating heterodimeric antibodies fromtwo different preexisting antibodies.

In one aspect, described herein is a heterodimeric antibody or fragmentthereof comprising one or more substitutions in each of the followingdomains: a first CH₃-domain, a second CH₃-domain, a CH₁-domain, aC_(L)-domain, a V_(H)-domain and a V_(L)-domain, wherein the one or moresubstitutions introduce charged amino acids that are electrostaticallyunfavorable to homodimer formation and electrostatically favorable toheterodimer formation.

In some variations, the first CH3-domain or the second CH3-domaincomprises an amino acid sequence differing from wild-type IgG amino acidsequence such that one or more positive-charged amino acids (e.g.,lysine, histidine and arginine) in the wild-type human IgG amino acidsequence are replaced with one or more negative-charged amino acids(e.g., aspartic acid and glutamic acid) at the corresponding position(s)in the CH3 domain. Alternatively, the first CH3-domain or the secondCH3-domain comprises an amino acid sequence differing from wild-type IgGamino acid sequence such that one or more negative-charged amino acidsin the wild-type human IgG amino acid sequence are replaced with one ormore positive-charged amino acids at the corresponding position(s) inthe CH3 domain.

In some variations, the CH1-domain or the CL-domain comprises an aminoacid sequence differing from wild-type IgG amino acid sequence such thatone or more positive-charged amino acids in wild-type IgG amino acidsequence are replaced with one or more negative-charged amino acids.Alternatively, the CH1-domain or the CL-domain comprises an amino acidsequence differing from wild-type IgG amino acid sequence such that oneor more negative-charged amino acids in wild-type IgG amino acidsequence are replaced with one or more positive-charged amino acids.

The VH-domain or the VL-domain of a heterodimeric antibody describedherein comprises, in some variations, an amino acid sequence differingfrom wild-type IgG amino acid sequence such that one or morepositive-charged amino acids in wild-type IgG amino acid sequence arereplaced with one or more negative-charged amino acids. Alternatively,the VH-domain or the VL-domain comprises an amino acid sequencediffering from wild-type IgG amino acid sequence such that one or morenegative-charged amino acids in wild-type IgG amino acid sequence arereplaced with one or more positive-charged amino acids.

In another aspect, described herein is a heterodimeric antibody orfragment thereof comprising a heavy chain comprising (a) a first aminoacid substitution at an AHo position selected from the group consistingof AHo positions 42-50 that introduces a charged amino acid at saidposition, (b) a second amino acid substitution at a position selectedfrom the group consisting of positions 126-213 (EU numbering) thatintroduces a charged amino acid at said position, (c) a third amino acidsubstitution at a position selected from the group consisting ofpositions 352-360 (EU numbering) that introduces a charged amino acid atsaid position, and (d) a fourth amino acid substitution at a positionselected from the group consisting of positions 395-403 (EU numbering)that introduces a charged amino acid, wherein the charged amino acid of(a) has the same charge as the charged amino acid of (b), and whereinthe charged amino acids of (c) and (d) have an opposite charge of thecharged amino acids of (a) and (b).

In some embodiments, the first amino acid substitution is at AHoposition 46, the second amino acid substitution is at EU position 183,the third amino acid substitution is at EU position 356 and the fourthamino acid substitution is at EU position 399. In some embodiments,glutamine at AHo position 46 is replaced with glutamic acid, serine atEU position 183 is replaced with glutamic acid, glutamic acid at EUposition 356 is replaced with lysine, and aspartic acid at EU position399 is replaced with lysine.

In another aspect, described herein is an antibody or fragment thereofcomprising a heavy chain comprising (a) a first amino acid substitutionat an AHo position selected from the group consisting of AHo positions35-43 that introduces a charged amino acid at said position, (b) asecond amino acid substitution at a position selected from the groupconsisting of positions 126-213 (EU numbering) that introduces a chargedamino acid at said position, (c) a third amino acid substitution at aposition selected from the group consisting of positions 388-398 (EUnumbering) that introduces a charged amino acid at said position, and(d) a fourth amino acid substitution at a position selected from thegroup consisting of positions 404-413 (EU numbering) that introduces acharged amino acid, wherein the charged amino acids of (c) and (d) havean opposite charge of the charged amino acids of (a) and (b). In someembodiments, the glutamine at AHo position 46 is replaced with lysine,serine at EU position 183 is replaced with lysine, lysine at EU position392 is replaced with aspartic acid and lysine at EU position 409 isreplaced with aspartic acid.

In yet another aspect, described herein is an antibody or fragmentcomprising a light chain comprising (a) a first amino acid substitutionat an AHo position selected from the group consisting of AHo positions42-50 that introduces a charged amino acid at said position, and (b) asecond amino acid substitution at a position selected from the groupconsisting of positions 126-213 (EU numbering) that introduces a chargedamino acid at said position. In some embodiments, glutamine at AHoposition 46 is replaced with glutamic acid and serine at EU position 176is replaced with glutamic acid.

In another aspect, described herein is an antibody comprising a heavychain comprising (a) a first amino acid substitution at an AHo positionselected from the group consisting of AHo positions 42-50 thatintroduces a charged amino acid at said position, (b) a second aminoacid substitution at a position selected from the group consisting ofpositions 126-213 (EU numbering) that introduces a charged amino acid atsaid position, (c) a third amino acid substitution at a positionselected from the group consisting of positions 352-360 (EU numbering)that introduces a charged amino acid at said position, and (d) a fourthamino acid substitution at a position selected from the group consistingof positions 395-403 (EU numbering) that introduces a charged aminoacid, wherein the charged amino acids of (a) and (b) have anegative-charge and the charged amino acids of (c) and (d) have apositive charge. In some embodiments, the first amino acid substitutionis at position AHo 46, the second amino acid substitution is at EUposition 183, the third amino acid substitution is at EU position 356and the fourth amino acid substitution is at EU position 399. In someembodiments, the antibody comprises a light chain comprisingpositive-charged amino acids at AHo position 46 and EU position 176.

In still another aspect, described herein is a heterodimeric antibodycomprising a first heavy chain and a second heavy chain and a firstlight chain and a second light chain, wherein the first heavy chaincomprises amino acid substitutions at AHo position 46 and EU positions183, 356 and 399, wherein the second heavy chain comprises amino acidsubstitutions at AHo position 46 and EU positions 183, 392 and 409, andwherein the first and second light chains comprise an amino acidsubstitution at AHo position 46 and EU position 176, wherein the aminoacid substitutions introduce a charged amino acid at said positions. Insome embodiments, the glutamine at position AHo 46 of the first heavychain is replaced with glutamic acid, the glutamine at position AHo 46of the second heavy chain is replaced with lysine, the glutamine atposition AHo 46 of the first light chain is replaced with lysine, theglutamine at position AHo 46 of the second light chain is replaced withglutamic acid, the serine at EU position 183 of the first heavy chain isreplaced with glutamic acid, the glutamic acid at EU position 356 of thefirst heavy chain is replaced with lysine, the glutamic acid at EUposition 399 of the first heavy chain is replaced with lysine, theserine at EU position 183 of the second heavy chain is replaced withlysine, the lysine at EU position 392 of the second heavy chain isreplaced with aspartic acid, and the lysine at EU position 409 of thesecond heavy chain is replaced with aspartic acid.

In some or any of the embodiments described herein, the antibody bindsto a region of sclerostin comprising amino acids 86-111 of SEQ ID NO: 1.

Also described herein is a heterodimeric antibody that binds to a regionof sclerostin comprising amino acids 86-111 of SEQ ID NO: 1, wherein theantibody comprises a first heavy chain and a first light chaincomprising a sclerostin-binding portion and a second heavy chain and asecond light chain comprising a DKK1-binding portion, wherein the firstheavy chain comprises amino acid substitutions at AHo position 46 and EUpositions 183, 356 and 399, wherein the second heavy chain comprisesamino acid substitutions at AHo position 46 and EU positions 183, 392and 409, wherein the first and second light chains comprise an aminoacid substitution at AHo position 46 and EU position 176, and whereinthe amino acid substitutions introduce a charged amino acid at saidpositions.

In yet another aspect, described herein is a heterodimeric antibody thatbinds to a region of sclerostin comprising amino acids 86-111 of SEQ IDNO: 1, the antibody comprising a first heavy chain and a first lightchain comprising a sclerostin-binding portion and a second heavy chainand a second light chain comprising a DKK1-binding portion, wherein thefirst heavy chain comprises amino acid substitutions at EU positions183, 356 and 399, wherein the second heavy chain comprises amino acidsubstitutions at EU positions 183, 392 and 409, and wherein the firstand second light chains comprise an amino acid substitution at EUposition 176, wherein the amino acid substitutions introduce a chargedamino acid at said positions.

Another aspect of the invention relates to a heterodimeric antibody thatbinds sclerostin and DKK-1, comprising a first heavy chain comprising aheavy chain variable region amino acid sequence of any one of thesclerostin antibodies described herein and comprising amino acidsubstitutions at EU positions 183, 356 and 399 of the first heavy chain,a second heavy chain comprising a heavy chain variable region amino acidsequence of any one of the DKK-1 antibodies described herein andcomprising amino acid substitutions at EU positions 183, 392 and 409 ofthe second heavy chain, a first light chain comprising a light chainvariable region amino acid sequence of any one of the sclerostinantibodies described herein and comprising an amino acid substitution atEU position 176 of the first light chain, and a second light chaincomprising a light chain variable region amino acid sequence of any ofthe DKK-1 antibodies described herein and comprising an amino acidsubstitution at EU position 176 of the second light chain; wherein theamino acid substitutions introduce a charged amino acid at saidpositions.

Another aspect of the invention relates to a heterodimeric antibody thatbinds sclerostin and DKK-1, comprising a first heavy chain comprising avariable region amino acid sequence selected from the group consistingof SEQ ID NOs: 378 and 366 and comprising amino acid substitutions at EUpositions 183, 356 and 399 of the first heavy chain, a second heavychain comprising a variable region amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1003 and 974 and comprising amino acidsubstitutions at EU positions 183, 392 and 409 of the second heavychain, a first light chain comprising a variable region amino acidsequence selected from the group consisting of SEQ ID NOs: 376 and 364and comprising an amino acid substitution at EU position 176 of thefirst light chain, and a second light chain comprising a variable regionamino acid sequence selected from the group consisting of SEQ ID NOs:1002 and 978 and comprising an amino acid substitution at EU position176 of the second light chain; wherein the amino acid substitutionsintroduce a charged amino acid at said positions.

In another aspect, described herein is an antibody that binds to aregion of sclerostin comprising amino acids 86-111 of SEQ ID NO: 1,wherein the antibody comprises substitutions in each of the followingdomains: a first CH3-domain, a second CH3-domain, a CH1-domain, and aCL-domain, wherein the one or more substitutions introduce charged aminoacids that are electrostatically unfavorable to homodimer formation andelectrostatically favorable to heterodimer formation.

Also described herein is an antibody that binds to a region ofsclerostin comprising amino acids 86-111 of SEQ ID NO: 1, wherein theantibody comprises a heavy chain having a CH3 domain comprising one ormore amino acid substitutions, wherein the one or more substitutionsintroduce charged amino acids that are electrostatically unfavorable tohomodimer formation and electrostatically favorable to heterodimerformation. In some embodiments, a negative charged amino acid in the CH3domain (e.g., at EU position D399, E356 or E357) is substituted with apositive charged amino acid. In some embodiments, amino acids at EUpositions D399, E356 and E357 are substituted with a positive chargedamino acid (e.g., lysine).

In alternative embodiments, a positive charged amino acid in the CH3domain (e.g., at EU position K370, K392 or K409) is substituted with anegative charged amino acid. In some embodiments, amino acids as EUpositions K370, K392 and K409 are substituted with a negative chargedamino acid (e.g., aspartic acid).

The heterodimeric antibody is, in some embodiments, an antibody, abispecific antibody, a monospecific monovalent antibody, a bispecificmaxibody, a monobody, a peptibody, a bispecific peptibody, a monovalentpeptibody or a receptor fusion protein.

Nucleic acids comprising a nucleotide sequence encoding any of theheterodimeric antibodies described herein are also provided, as well asvectors and host cells comprising the nucleic acid (or vector).

Another aspect of the invention relates to method of increasing bonemineral density in a mammalian subject comprising administering aheterodimeric antibody described herein to the subject in an amounteffective to increase bone mineral density in the subject. The inventionalso includes methods of using heterodimeric antibodies described hereinfor increasing bone mineral density. Methods of using as describedherein can alternatively be characterized as uses of the heterodimericantibodies for increasing bone mineral density.

The invention also includes compositions comprising a heterodimericantibody described herein and a pharmaceutically acceptable carrier,diluent or adjuvant. In some embodiments, less than 5% (or less than 4%,or less than 3%, or less than 2% or less than 1% or less) of theantibody in the composition is in aggregate form after two weeks ofstorage at about 4° C. The amount of antibody aggregation in thecomposition can be determined, for example, by Size SeclusionChromatography (SEC) or Dynamic Light Scattering (DLS).

In another aspect, described herein is a composition comprising aheterodimeric antibody or fragment thereof and a pharmaceuticallyacceptable carrier, diluent or adjuvant, the heterodimeric antibody orfragment thereof comprising one or more substitutions in each of thefollowing domains: a first CH₃-domain, a second CH₃-domain, aCH₁-domain, a C_(L)-domain, a V_(H)-domain and a V_(L)-domain, whereinthe one or more substitutions introduce charged amino acids that areelectrostatically unfavorable to homodimer formation andelectrostatically favorable to heterodimer formation; wherein less than5% of the antibody or fragment in the composition is in aggregate formafter two weeks of storage at about 4° C.

In another aspect, described herein is a composition comprising aheterodimeric antibody that binds to a region of sclerostin comprisingamino acids 86-111 of SEQ ID NO: 1 and a pharmaceutically acceptablecarrier, diluent or adjuvant, the antibody comprising substitutions ineach of the following domains: a first CH₃-domain, a second CH₃-domain,a CH₁-domain, and a C_(L)-domain, wherein the one or more substitutionsintroduce charged amino acids that are electrostatically unfavorable tohomodimer formation and electrostatically favorable to heterodimerformation; wherein less than 5% of the antibody in the composition is inaggregate form after two weeks of storage at about 4° C.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited within the body of this specification are expresslyincorporated by reference in their entirety.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, tissue culture and transformation, protein purification, etc.Enzymatic reactions and purification techniques may be performedaccording to the manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The following proceduresand techniques may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thespecification. See, e.g., Sambrook et al., 2001, Molecular Cloning: ALaboratory Manuel, 3rd ed., Cold Spring Harbor Laboratory Press, coldSpring Harbor, N.Y., which is incorporated herein by reference for anypurpose. Unless specific definitions are provided, the nomenclature usedin connection with, and the laboratory procedures and techniques of,analytic chemistry, organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, chemicalanalyses, pharmaceutical preparation, formulation, and delivery andtreatment of patients.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the in vivo study design for the followingheterodimeric antibodies: (1) Ab23-Ab6.147 v2, (2) Ab5-Ab6.37.5 v1, and(3) Ab5-Ab6.147.

FIG. 2 compares percentage of bone mass density (BMD) increase in lumbarvertebrae and leg in mice between monospecific Ig (Ab5), bispecific DVD(6.147-Ab23) and heterodimeric antibodies (1, 2, & 3) at week 3.

FIGS. 3A-3D show the pharmacokinetic (PK) profiles of foursclerostin-DKK1 heterodimeric antibodies (i.e., Ab23-6.37.5 v1,Ab5-6.37.5 v1, Ab5-6.147 v2 and Ab23-6.147 v2).

FIGS. 4A-4F: Configurations of bispecific antibody variants usingdifferent approaches (a) A pair of charged residues K/D in variableregions binding to antigen A combined with a pair of charged residuesD/K in C_(H)1/CL to enforce the LC to pair with its cognate HC; A pairof charged residues D/K in variable regions binding to antigen Bcombined with a pair of charged residues K/D in C_(H)1/CL to enforce theLC to pair with its cognate HC. The heterodimerizing charge residues inC_(H)3 domains are also indicated. (b) A pair of charged residues D/K invariable regions binding to antigen A combined with two pairs of chargedresidues KK/DD in C_(H)1/CL to enforce the LC to pair with its cognateHC; A pair of charged residues K/D in variable regions binding toantigen B combined with two pairs of charged residues DD/KK in C_(H)1/CLto enforce the LC to pair with its cognate HC. The charge residues forheterodimerization in C_(H)3 domains are also indicated. (c) Two pairsof charged residues KK/DD in variable regions binding to antigen Acombined with one pair of charged residues D/K in C_(H)1/CL to enforcethe LC to pair with its cognate HC; two pairs of charged residues DD/KKin variable regions binding to antigen B combined with one pair ofcharged residues K/D in C_(H)1/CL to enforce the LC to pair with itscognate HC. The charge residues for heterodimerization in C_(H)3 domainsare also indicated. (d) Two pairs of charged residues KK/DD in variableregions binding to antigen A combined with two pairs of charged residuesDD/KK in C_(H)1/CL to enforce the LC to pair with its cognate HC; twopairs of charged residues DD/KK in variable regions binding to antigen Bcombined with two pair of charged residues KK/DD in C_(H)1/CL to enforcethe LC to pair with its cognate HC. The charge residues forheterodimerization in C_(H)3 domains are also indicated. (e) One pair ofcharged residues K/D and one pair of cysteine residues in variableregions binding to antigen A combined with one pair of charged residuesD/K in C_(H)1/CL to enforce the LC to pair with its cognate HC; One pairof cysteine residues and one pair of charged residues D/K in variableregions binding to antigen B combined with one pair of charged residuesK/D in C_(H)1/CL to enforce the LC to pair with its cognate HC. Thecharge residues for heterodimerization in C_(H)3 domains are alsoindicated. © represents the cysteine residue, a disulfide bond formedbetween two cysteine residues could stabilize the Fab region withcorrect LC/HC pairing. (f) One pair of charged residues K/D and one pairof small/bulky residues in variable regions binding to antigen Acombined with one pair of charged residues D/K in C_(H)1/CL to enforcethe LC to pair with its cognate HC; one pair of charged residues D/K andone pair of bulky/small residues and in variable regions binding toantigen B combined with one pair of charged residues K/D in C_(H)1/CL toenforce the LC to pair with its cognate HC. The charge residues forheterodimerization in C_(H)3 domains are also indicated. Protrusivetriangle represents the bulky residue; recessive triangle represents thesmall residue. A knob-into-hole effect may work cooperatively withelectrostatic steering effect to guide and stabilize the correct LC/HCpairing.

FIGS. 5A-5B: Alignment of human heavy chain V and J regions. Numberingis based on the AHo system. Interface residues are highlighted.

FIGS. 6A-6B: Alignment of human kappa chain V and J regions. Numberingis based on the AHo system. Interface residues are highlighted.

FIGS. 7A-7B: Alignment of human lambda chain V and J regions. Numberingis based on the AHo system. Interface residues are highlighted.

DETAILED DESCRIPTION OF THE INVENTION

The present application is based on the discovery that heterodimericIgGs could be produced by engineering the heavy chain and light chain ofthe two different antibodies in such a way that they can assembleexclusively into a heterodimeric antibody without other contaminatingspecies. In one aspect, heterodimeric pairing is achieved by engineeringthe CH3 regions of two heavy chains so that it forms a heterodimerexclusively. Further, electrostatic steering achieved by engineeringinterface residues between the light chains (LC) and the heavy chains(HC) prevents mis-pairing of light chains to the non-cognate heavychains when two different heavy chain and light chain pairs areassembling to form a desired four-chain heterodimeric antibody. Asdescribed herein, an exemplary strategy comprises introducing one ormore negatively-charged residues (e.g., Asp or Glu) in a first lightchain (LC1) and one or more positively-charged residues (e.g., Lys, Hisor Arg) in the companion heavy chain (HC1) at the LC 1/HC 1 interfacewhile introducing one or more positively-charged residues (e.g., Lys,His or Arg) in a second light chain (LC2) and one or morenegatively-charged residues (e.g., Asp or Glu) in the companion heavychain (HC2) at the LC2/HC2 interface. The electrostatic steering effectdirects the LC1 to pair with HCl and LC2 to pair with HC2, as theopposite charged residues (polarity) at the interface attract, while thesame type of charged residues (polarity) at an interface causesrepulsion, resulting in suppression of the unwanted HC/LC pairings.

The LC/HC interface residues selected for engineering are buried andspatially close within the VL/VH and CL/CH1 interfaces. The targetresidues are well conserved among different antibody families. Otherways of engineering the light and heavy chains to form specificheterodimers include replacing one pair of charged residues in the VL/VHinterface with a pair of cysteine residues to form a disulfide bond tostabilize the Fab region, replacing one or more hydrophilic residues(e.g., glycine) in the VL/VH interface with a hydrophobic residue (e.g.,glutamine), or engineering a pair of bulky/small residues at the VL/VHinterface to exert a knob-into-hole effect to accommodate the correctLC/HC pairing. The strategy described herein can be used to efficientlyproduce a full-length heterodimeric antibody from two preexistingantibodies without using artificial linkers. The resulting heterodimericantibodies are stable and amenable to commercial manufacturing withoutexcessive aggregation or loss of yield. Because this new version ofheterodimeric antibody can target two different antigens or twodifferent epitopes on the same antigen simultaneously, it may havesignificant potential to uniquely treat many diseases.

The term “interface” as used herein refers to the association surfacethat results from interaction one or more amino acids in a firstantibody domain with one or more amino acids of a second antibodydomain. Exemplary interfaces include, CH1/CL, VH/VL and CH3/CH3. In someembodiments, the interface includes, for example, hydrogen bonds,electrostatic interactions, or salt bridges between the amino acidsforming an interface.

In one aspect, described herein is a heterodimeric antibody or fragmentthereof comprising one or more substitutions in each of the followingdomains: a first CH₃-domain, a second CH₃-domain, a CH₁-domain, aC_(L)-domain, a V_(H)-domain and a V_(L)-domain, wherein the one or moresubstitutions introduce charged amino acids that are electrostaticallyunfavorable to homodimer formation and electrostatically favorable toheterodimer formation.

Heterodimeric antibodies described herein can comprise any constantregion. The light chain constant region can be, for example, a kappa- orlambda-type light chain constant region, e.g., a human kappa- orlambda-type light chain constant region. The heavy chain constant regioncan be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-typeheavy chain constant region, e.g., a human alpha-, delta-, epsilon-,gamma-, or mu-type heavy chain constant region. In one embodiment thelight or heavy chain constant region is a fragment, derivative, variant,or mutein of a naturally-occurring constant region.

In some variations, the first CH3-domain or the second CH3-domaincomprises an amino acid sequence differing from wild-type IgG amino acidsequence such that one or more positive-charged amino acids (e.g.,lysine, histidine and arginine) in the wild-type human IgG amino acidsequence are replaced with one or more negative-charged amino acids(e.g., aspartic acid and glutamic acid) at the corresponding position(s)in the CH3 domain. Alternatively, the first CH3-domain or the secondCH3-domain comprises an amino acid sequence differing from wild-type IgGamino acid sequence such that one or more negative-charged amino acidsin the wild-type human IgG amino acid sequence are replaced with one ormore positive-charged amino acids at the corresponding position(s) inthe CH3 domain.

In some variations, the CH1-domain or the CL-domain comprises an aminoacid sequence differing from wild-type IgG amino acid sequence such thatone or more positive-charged amino acids in wild-type IgG amino acidsequence are replaced with one or more negative-charged amino acids.Alternatively, the CH1-domain or the CL-domain comprises an amino acidsequence differing from wild-type IgG amino acid sequence such that oneor more negative-charged amino acids in wild-type IgG amino acidsequence are replaced with one or more positive-charged amino acids.

The VH-domain or the VL-domain of a heterodimeric antibody describedherein comprises, in some variations, an amino acid sequence differingfrom wild-type IgG amino acid sequence such that one or morepositive-charged amino acids in wild-type IgG amino acid sequence arereplaced with one or more negative-charged amino acids. Alternatively,the VH-domain or the VL-domain comprises an amino acid sequencediffering from wild-type IgG amino acid sequence such that one or morenegative-charged amino acids in wild-type IgG amino acid sequence arereplaced with one or more positive-charged amino acids.

In another aspect, described herein is a heterodimeric antibody orfragment thereof comprising a heavy chain comprising (a) a first aminoacid substitution at an AHo position selected from the group consistingof AHo positions 42-50 that introduces a charged amino acid at saidposition, (b) a second amino acid substitution at a position selectedfrom the group consisting of positions 126-213 (EU numbering) thatintroduces a charged amino acid at said position, (c) a third amino acidsubstitution at a position selected from the group consisting ofpositions 352-360 (EU numbering) that introduces a charged amino acid atsaid position, and (d) a fourth amino acid substitution at a positionselected from the group consisting of positions 395-403 (EU numbering)that introduces a charged amino acid, wherein the charged amino acid of(a) has the same charge as the charged amino acid of (b), and whereinthe charged amino acids of (c) and (d) have an opposite charge of thecharged amino acids of (a) and (b).

Herein, the position of particular amino acids within the frameworkregions of the variable domains (described below) is described using theAHo numbering system. Because antibody CDR amino acid sequence lengthvaries from antibody to antibody, numbering residues based on the linearsequence (assuming the first residue as position 1) leads to frameworkresidues having different position numbers between antibodies. UsingKabat or EU numbering scheme could avoid this conflict and enablecomparison of framework positions across antibodies. However,structurally equivalent positions can have a different Kabat or EUnumber. Similarly, residues having same Kabat number can be present attwo different locations on the structure. Annemarie Honegger and AndreasPluckthun developed a structure based numbering scheme (AHo), whichintroduces gaps in the CDR regions to minimize deviation from theaverage structure of the aligned domains. (Honegger, A., and Plückthun,A. (2001). J. Mol. Biol. 309, 657-670) This leads to structurallyequivalent positions having the same residue number when two differentantibodies are compared. This enables comparison of the effect ofsubstitutions in the variable domain framework region betweenantibodies. FIGS. 5-7 provide the AHo numbering for human heavy chain,kappa, and lambda variable domain regions, respectively.

As used herein, the term “framework” or “framework sequence” refers tothe region or sequence of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, -L2,and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) alsodivide the framework regions on the light chain and the heavy chain intofour sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 ispositioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3between FR3 and FR4. Without specifying the particular sub-regions asFR1, FR2, FR3 or FR4, a framework region, represents the combined FR'swithin the variable region of a single, naturally occurringimmunoglobulin chain. As used herein, a FR represents one of the foursub-regions, and FRs represents two or more of the four sub-regionsconstituting a framework region.

“Substituting” or “substitution of” an amino acid refers to substitutingthe original amino acid residue for one or more other amino acidresidue(s).

Heavy Chain Modifications

To maximize efficiency of a particular heavy chain binding to itscognate light chain, both the heavy and light chains containcomplimentary amino acid substitutions. By “complimentary amino acidsubstitutions,” it is meant that a substitution to a positive-chargeamino acid in the heavy chain is paired with a negative-charged aminoacid substitution to an amino acid in the light chain that associateswith the heavy chain residue. Likewise, a substitution to anegative-charge amino acid in the heavy chain is paired with apositive-charged amino acid substitution to an amino acid in the lightchain that associates with the heavy chain residue.

In some embodiments, an antibody heavy chain variable region isengineered. FIG. 5 is a germline alignment of human heavy chain variabledomain V and J regions. In some embodiments, the heterodimeric antibodycomprises a heavy chain variable region that is at least about 70%, atleast about 71%, at least about 72%, at least about 73%, at least about74%, at least about 75%, at least about 76%, at least about 77%, atleast about 78%, at least about 79%, at least about 80%, at least about81%, at least about 82%, at least about 83%, at least about 84%, atleast about 85%, at least about 86%, at least about 87%, at least about88%, at least about 89%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% identical to a human germline heavy chain variableregion.

V region interface residues (i.e., amino acid residues that mediateassembly of the VH and VL domains) within the VH domain include AHopositions 1 (Kabat position 1), 3 (Kabat position 3), 42 (Kabat position35), 44 (Kabat position 37), 46 (Kabat position 39), 50 (Kabat position43), 51 (Kabat position 44), 52 (Kabat position 45), 53 (Kabat position46), 54 (Kabat position 47), 57 (Kabat position 50), 70 (Kabat position59), 103 (Kabat position 89), 105 (Kabat position 91), and 107 (Kabatposition 93). J region interface residues within the include AHopositions 139 (Kabat position 103), 140 (Kabat position 104), 141 (Kabatposition 105), and 142 (Kabat position 106). In some embodiments, one ormore interface residues are substituted with a charged (positive- ornegative-charged) amino acid.

In some embodiments, the amino acid at AHo position 46 (Kabat position39) of the VH domain is replaced with a positive-charged amino acid. Inalternative embodiments, the amino acid at AHo position 46 (Kabatposition 39) of the VH domain is replaced with a negative-charged aminoacid.

In some embodiments, the amino acid at AHo position 51 (Kabat position44) and/or AHo position 141 (Kabat position 105) of the VH domain arereplaced with a charged amino acid. In some embodiments, the amino acidat AHo position 51 is substituted for a positive-charged amino acid,e.g., lysine. In alternative embodiments, the amino acid at AHo position51 is substituted for a negative-charged amino acid, e.g., asparticacid. In some embodiments, the amino acid at AHo position 141 issubstituted for a positive-charged amino acid, e.g., lysine. Inalternative embodiments, the amino acid at AHo position 141 issubstituted for a negative-charged amino acid, e.g., aspartic acid. Insome embodiments, the amino acid at AHo positions 51 and AHo position141 are substituted for a positive-charged amino acid, e.g., lysine, ora negative charged amino acid, e.g., aspartic acid. Such embodiments mayfurther comprise a substitution at AHo position 46 to a positive- ornegative-charged amino acid.

The CH1 region of the heavy chain also complexes with the light chain,and this region can be engineered to increase the efficiency of aparticular heavy chain pairing with its cognate light chain. Assemblymay be facilitated by introducing a cysteine residue into the heavy andlight chain at or near the interface to allow formation of di-sulfidebonds, altering amino acids to create a knobs-into-holes effect, andelectros engineering similar to that described herein for the variableregions.

In some embodiments, the heterodimeric antibody comprises a heavy chainCH1 region that is at least about 70%, at least about 71%, at leastabout 72%, at least about 73%, at least about 74%, at least about 75%,at least about 76%, at least about 77%, at least about 78%, at leastabout 79%, at least about 80%, at least about 81%, at least about 82%,at least about 83%, at least about 84%, at least about 85%, at leastabout 86%, at least about 87%, at least about 88%, at least about 89%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99% identicalto a human germline heavy chain CH1 region.

In some embodiments, one or more amino acids in the CH1 domain of theheterodimeric antibody at an EU position selected from the groupconsisting of F126, P127, L128, A141, L145, K147, D148, H168, F170,P171, V173, Q175, S176, S183, V185 and K213 is/are replaced with acharged amino acid. In this regard, a particularly preferred residue forsubstitution with a negative- or positive-charged amino acid is S183 (EUnumbering system). In some embodiments, S183 is substituted with apositive-charged amino acid. In alternative embodiments, S183 issubstituted with a negative-charged amino acid.

The heterodimeric antibody described herein optionally further comprisestwo CH3 domains, at least one of which contains one or moresubstitutions introducing a non-native charged amino acid into thedomain. In some embodiments, each CH3 domain comprises one or more aminoacid substitutions in the CH3 domain to disfavor homodimerization, andmore preferably, favor heterodimerization with the corresponding CH3domain. International Publication No. WO 2009/089004 (incorporatedherein by reference in its entirety) describes compositions and methodsfor engineering the CH3 domain interface to decrease homodimerizationand increase heterodimerization between two CH3-domain-containingmolecules. In some embodiments, amino acids at one or more positionsselected from the group consisting of 399, 356 and 357 (EU numberingsystem) of the CH3 domain are replaced with a negative-charged aminoacid. In some embodiments, amino acids at one or more positions selectedfrom the group consisting of 370, 392 and 409 (EU numbering system) arereplaced with a positive-charged amino acid. In alternative embodiments,amino acids at one or more positions selected from the group consistingof 399, 356 and 357 (EU numbering system) of the CH3 domain are replacedwith a positive-charged amino acid. In further embodiments, amino acidsat one or more positions selected from the group 370, 392 and 409 (EUnumbering system) are replaced with a negative-charged amino acid. Insome embodiments, the heterodimeric antibody comprises a first heavychain comprising positive-charged amino acid at positions 399 and 356(e.g., D399K and E356K), and a second heavy chain comprisingnegative-charged amino acids at positions 392 and 409 (e.g., K392D andK409D).

In one aspect, a heterodimeric antibody described herein comprises (a) afirst amino acid substitution at an AHo position selected from the groupconsisting of AHo positions 42-50 that introduces a charged amino acidat said position, (b) a second amino acid substitution at an EU positionselected from the group consisting of EU positions 126-213 thatintroduces a charged amino acid at said position, (c) a third amino acidsubstitution at an EU position selected from the group consisting of EUpositions 352-360 that introduces a charged amino acid at said position,and (d) a fourth amino acid substitution at an EU position selected fromthe group consisting of EU positions 395-403 that introduces a chargedamino acid, wherein the charged amino acid of (a) has the same charge asthe charged amino acid of (b), and wherein the charged amino acids of(c) and (d) have an opposite charge of the charged amino acids of (a)and (b). For example, in some embodiments, the charged amino acids of(a) and (b) have a positive-charge and the charged amino acids of (c)and (d) have a negative-charge. In alternative embodiments, the chargedamino acids of (a) and (b) have a negative-charge and the charged aminoacids of (c) and (d) have a positive-charge. In some embodiments, thefirst amino acid substitution is at position AHo 46, the second aminoacid substitution is at EU position 183, the third amino acidsubstitution is at EU position 356 and the fourth amino acidsubstitution is at EU position 399.

In another aspect, a heterodimeric antibody described herein comprises aheavy chain comprising (a) a first amino acid substitution at an AHoposition selected from the group consisting of AHo positions 42-50 thatintroduces a charged amino acid at said position, (b) a second aminoacid substitution at an EU position selected from the group consistingof EU positions 126-213 that introduces a charged amino acid at saidposition, (c) a third amino acid substitution at an EU position selectedfrom the group consisting of EU positions 388-397 that introduces acharged amino acid at said position, and (d) a fourth amino acidsubstitution at an EU position selected from the group consisting of EUpositions 404-413 that introduces a charged amino acid, wherein thecharged amino acid of (a) has the same charge as the charged amino acidof (b), and wherein the charged amino acids of (c) and (d) have anopposite charge of the charged amino acids of (a) and (b). For example,in some embodiments, the charged amino acids of (a) and (b) have apositive-charge and the charged amino acids of (c) and (d) have anegative-charge. In alternative embodiments, the charged amino acids of(a) and (b) have a negative-charge and the charged amino acids of (c)and (d) have a positive-charge. In some embodiments, the first aminoacid substitution is at AHo position 46, the second amino acidsubstitution is at EU position 183, the third amino acid substitution isat EU position 392 and the fourth amino acid substitution is at EUposition 409.

Also provided herein is a heterodimeric antibody comprising a firstheavy chain and a second heavy chain and a first light chain and asecond light chain, wherein the first heavy chain comprises amino acidsubstitutions at positions 46 (AHo, Kabat 39), 183 (EU), 356 (EU) and399 (EU), wherein the second heavy chain comprises amino acidsubstitutions at positions 46 (AHo), 183 (EU), 392 (EU) and 409 (EU),and wherein the first and second light chains comprise an amino acidsubstitution at positions 46 (AHo, Kabat 38) and 176 (EU), wherein theamino acid substitutions introduce a charged amino acid at saidpositions. In some embodiments, the glutamine at position AHo 46 (Kabat39) of the first heavy chain is replaced with glutamic acid, theglutamine at position AHo 46 (Kabat 39) of the second heavy chain isreplaced with lysine, the glutamine at position AHo 46 (Kabat 38) of thefirst light chain is replaced with lysine, the glutamine at position AHo46 (Kabat 38) of the second light chain is replaced with glutamic acid,the serine at position 183 (EU) of the first heavy chain is replacedwith glutamic acid, the glutamic acid at position 356 (EU) of the firstheavy chain is replaced with lysine, the glutamic acid at position 399(EU) of the first heavy chain is replaced with lysine, the serine atposition 183 (EU) of the second heavy chain is replaced with lysine, thelysine at position 392 (EU) of the second heavy chain is replaced withaspartic acid, and/or the lysine at position 409 (EU) of the secondheavy chain is replaced with aspartic acid.

In another aspect, described herein is an antibody that binds to aregion of sclerostin comprising amino acids 86-111 of SEQ ID NO: 1,wherein the antibody comprises a heavy chain having a CH3 domaincomprising one or more amino acid substitutions, wherein the one or moresubstitutions introduce charged amino acids that are electrostaticallyunfavorable to homodimer formation and electrostatically favorable toheterodimer formation. In some embodiments, a negative charged aminoacid in the CH3 domain (e.g., at EU position D399, E356 or E357) issubstituted with a positive charged amino acid. In some embodiments,amino acids at EU positions D399, E356 and E357 are substituted with apositive charged amino acid (e.g., lysine). International PublicationNo. WO 2009/089004 (incorporated herein by reference in its entirety)describes compositions and methods for engineering the CH3 domaininterface to decrease homodimerization and increase heterodimerizationbetween two CH3-domain-containing molecules.

Light Chain Modifications

As discussed above, to maximize binding of a particular heavy chain toits cognate light chain, both the heavy and light chains preferablycontain complimentary amino acid substitutions to electrostaticallysteer the chains to assemble. Thus, in various embodiments, a lightchain comprises one or more amino acid substitutions that compliment aheavy chain substitution discussed above.

In some embodiments, the light chain is a kappa light chain. Figure. 6is a germline alignment of human kappa light chain variable domain V andJ regions. In some embodiments, the heterodimeric antibody comprises akappa chain variable region that is at least about 70%, at least about71%, at least about 72%, at least about 73%, at least about 74%, atleast about 75%, at least about 76%, at least about 77%, at least about78%, at least about 79%, at least about 80%, at least about 81%, atleast about 82%, at least about 83%, at least about 84%, at least about85%, at least about 86%, at least about 87%, at least about 88%, atleast about 89%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% identical to a human germline kappa chain variable region.

V region interface residues (i.e., amino acid residues that mediateassembly of the VH and VL domains) within the VL domain include AHopositions 40 (Kabat 32), 42 (Kabat 34), 43 (Kabat 35), 44 (Kabat 36), 46(Kabat 38), 49 (Kabat 41), 50 (Kabat 42), 51 (Kabat 43), 52 (Kabat 44),53 (Kabat 45), 54 (Kabat 46), 56 (Kabat 48), 57 (Kabat 49), 58 (Kabat50), 67 (Kabat 51), 69 (Kabat 53), 70 (Kabat 54), 71 (Kabat 55), 72(Kabat 56), 73 (Kabat 57), 74 (Kabat 58), 103 (Kabat 85), 105 (Kabat87), 107 (Kabat 89), 108 (Kabat 90), and 109 (Kabat 91). J regionresidues include AHo positions 116, 117, and 118. In some embodiments,one or more interface residues in the VL domain are substituted with acharged amino acid, preferably that has an opposite charge to thoseintroduces into the cognate heavy chain variable domain (i.e., the VHdomain). In some embodiments, the amino acid at AHo position 46 (Kabat38) of the VL domain is replaced with a positive-charged amino acid. Insome embodiments, such as when the amino acid at AHo position 46 (Kabat39) in the VH domain is substituted with a positive-charged amino acid),the amino acid at AHo position 46 (Kabat 38) of the VL domain isreplaced with a negative-charged amino acid.

In some embodiments, the amino acid at AHo positions 51 (Kabat 43)and/or AHo position 141 (Kabat 100) are substituted for a positive- ornegative-charged amino acid. Such embodiments may further include havingthe amino acid at AHo position 46 substituted for a positive- ornegative-charged amino acid. In some embodiments, the amino acid at AHoposition 51 is substituted for a positive-charged amino acid, e.g.,lysine. In alternative embodiments, the amino acid at AHo position 51 issubstituted for a negative-charged amino acid, e.g., aspartic acid. Insome embodiments, the amino acid at AHo position 141 is substituted fora positive-charged amino acid, e.g., lysine. In alternative embodiments,the amino acid at AHo position 141 is substituted for a negative-chargedamino acid, e.g., aspartic acid. In some embodiments, the amino acid atAHo position 51 and AHo position 141 are substituted for apositive-charged amino acid, e.g., lysine, or a negative-charged aminoacid, e.g., aspartic acid. Such embodiments may further comprise asubstitution at AHo position 46 (Kabat 38) to a positive- ornegative-charged amino acid.

The constant region of the light chain (i.e., the CL domain) alsocomplexes with the heavy chain, and this region can be engineered toincrease the efficiency of a particular light chain pairing with itscognate heavy chain. Assembly may be facilitated by introducing acysteine residue into the heavy and light chain at or near the interfaceto allow formation of di-sulfide bonds, altering amino acids to create aknobs-into-holes effect, and electrostatic engineering similar to thatdescribed herein for the variable regions.

In embodiments where the light chain, is a kappa light chain, one ormore amino acids in the CL domain of the heterodimeric antibody at aposition (EU and Kabat numbering in a kappa light chain) selected fromthe group consisting of F116, F118, S121, D122, E123, Q124, S131, V133,L135, N137, N138, Q160, S162, T164, S174 and S176 are replaced with acharged amino acid. In some embodiments, an exemplary residue forsubstitution with a negative- or positive-charged amino acid is theamino acid at position 176 (EU and Kabat numbering system) of the CLdomain. In some embodiments, the amino acid at position 176 of the CLdomain is replaced with a positive-charged amino acid. In alternativeembodiments, the amino acid at position 176 of the CL domain is replacedwith a negative-charged amino acid, e.g., aspartic acid.

In some embodiments, the light chain is a lambda light chain. In someembodiments, one or more amino acids in the CL domain of theheterodimeric antibody at a position (Kabat numbering in a lambda chain)selected from the group consisting of T116, F118, S121, E123, E124,K129, T131, V133, L135, S137, E160, T162, S165, Q167, A174, S176 andY178 are replaced with a charged amino acid. In some embodiments, anexemplary residue for substitution with a negative- or positive-chargedamino acid is the amino acid at position 176 (EU and Kabat numberingsystem) of the CL domain. In some embodiments, the amino acid atposition 176 of the CL domain is replaced with a positive-charged aminoacid. In alternative embodiments, the amino acid at position 176 of theCL domain is replaced with a negative-charged amino acid, e.g., asparticacid.

FIG. 7 is a germline alignment of human lambda light chain variabledomain V and J regions. In some embodiments, the antigen binding proteinor antibody comprises a light chain variable region that is at leastabout 70%, at least about 71%, at least about 72%, at least about 73%,at least about 74%, at least about 75%, at least about 76%, at leastabout 77%, at least about 78%, at least about 79%, at least about 80%,at least about 81%, at least about 82%, at least about 83%, at leastabout 84%, at least about 85%, at least about 86%, at least about 87%,at least about 88%, at least about 89%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99% identical to a human germline lambdachain variable region.

V region interface residues of the human germline lambda chain variableregion in the CL domain include AHo positions 40 (Kabat 32), 42 (Kabat34), 43 (Kabat 35), 44 (Kabat 36), 46 (Kabat 38), 49 (Kabat 41), 50(Kabat 42), 51 (Kabat 43), 52 (Kabat 44), 53 (Kabat 45), 54 (Kabat 46),56 (Kabat 48), 57 (Kabat 49), 58 (Kabat 50), 67 (Kabat 51), 69 (Kabat53), 70 (Kabat 54), 71 (Kabat 55), 72 (Kabat 56), 73 (Kabat 57), 74(Kabat 58), 103 (Kabat 85), 105 (Kabat 87), 107 (Kabat 89), 108 (Kabat90), and 109 (Kabat 91). J region residues include AHo positions 139 and140. The substitution of one or more of the amino acids at thesepositions with a charged amino acid is contemplated. In preferredembodiments, the one or more amino acids is substituted with a positive-or negative-charged amino acid, which is an opposite charge to thoseintroduce into the cognate heavy chain variable domain.

In some embodiments, the amino acid at AHo positions 51 (Kabat 43)and/or AHo position 141 (Kabat 100) of the lambda variable region aresubstituted for a positive- or negative-charged amino acid. Suchembodiments may further include having the amino acid at AHo position 46substituted for a positive- or negative-charged amino acid. In someembodiments, the amino acid at AHo position 51 is substituted for apositive-charged amino acid, e.g., lysine. In alternative embodiments,the amino acid at AHo position 51 is substituted for a negative-chargedamino acid, e.g., aspartic acid. In some embodiments, the amino acid atAHo position 141 is substituted for a positive-charged amino acid, e.g.,lysine. In alternative embodiments, the amino acid at AHo position 141is substituted for a negative-charged amino acid, e.g., aspartic acid.In some embodiments, the amino acid at AHo position 51 and AHo position141 are substituted for a positive-charged amino acid, e.g., lysine, ora negative-charged amino acid, e.g., aspartic acid. Such embodiments mayfurther comprise a substitution at AHo position 46 to a positive- ornegative-charged amino acid.

Optional Further Modifications

The heavy chains of the heterodimeric antibodies described herein mayfurther comprise one of more mutations that affect binding of theantibody containing the heavy chains to one or more Fc receptors. One ofthe functions of the Fc portion of an antibody is to communicate to theimmune system when the antibody binds its target. This is commonlyreferenced as “effector function.” Communication leads toantibody-dependent cellular cytotoxicity (ADCC), antibody-dependentcellular phagocytosis (ADCP), and/or complement dependent cytotoxicity(CDC). ADCC and ADCP are mediated through the binding of the Fc to Fcreceptors on the surface of cells of the immune system. CDC is mediatedthrough the binding of the Fc with proteins of the complement system,e.g., C1q.

The IgG subclasses vary in their ability to mediate effector functions.For example, IgG1 is superior to IgG2 and IgG4 at mediating ADCC andCDC. The effector function of an antibody can be increased, ordecreased, by introducing one or more mutations into the Fc. Embodimentsof the invention include heterodimeric antibodies, having an Fcengineered to increase effector function (U.S. Pat. No. 7,317,091 andStrohl, Curr. Opin. Biotech., 20:685-691, 2009; both incorporated hereinby reference in its entirety). Exemplary IgG1 Fc molecules havingincreased effector function include those having one or more of thefollowing substitutions [numbering based on the EU numbering scheme]:

S239D/I332E S239D/A330S/I332E S239D/A330L/I332E S298A/D333A/K334AP247I/A339D P247I/A339Q D280H/K290S D280H/K290S/S298D D280H/K290S/S298VF243L/R292P/Y300L F243L/R292P/Y300L/P396L F243L/R292P/Y300L/V305I/P396LG236A/S239D/I332E K326A/E333A K326W/E333S K290E/S298G/T299AK290N/S298G/T299A K290E/S298G/T299A/K326E K290N/S298G/T299A/K326E K334VL235S+S239D+K334V Q311M+K334V S239D+K334V F243V+K334V E294L+K334VS298T+K334V E233L+Q311M+K334V L234I+Q311M+K334V S298T+K334V A330M+K334VA330F+K334V Q311M+A330M+K334V Q311M+A330F+K334V S298T+A330M+K334VS298T+A330F+K334V S239D+A330M+K334V S239D+S298T+K334V L234Y+K290Y+Y296WL234Y+F243V+Y296W L234Y+E294L+Y296W L234Y+Y296W K290Y+Y296W

Further embodiments of the invention include heterodimeric antibodies,having an Fc engineered to decrease effector function. Exemplary Fcmolecules having decreased effector function include those having one ormore of the following substitutions [numbering based on the EU numberingscheme]:

N297A (IgG1) L234A/L235A (IgG1) V234A/G237A (IgG2) L235A/G237A/E318A(IgG4) H268Q/V309L/A330S/A331S (IgG2) C220S/C226S/C229S/P238S (IgG1)C226S/C229S/E233P/L234V/L235A (IgG1) L234F/L235E/P331S (IgG1)S267E/L328F (IgG1)

Another method of increasing effector function of IgG Fc-containingproteins is by reducing the fucosylation of the Fc. Removal of the corefucose from the biantennary complex-type oligosaccharides attached tothe Fc greatly increased ADCC effector function without altering antigenbinding or CDC effector function. Several methods are known for reducingor abolishing fucosylation of Fc-containing molecules, e.g., antibodies.These include recombinant expression in certain mammalian cell linesincluding a FUT8 knockout cell line, variant CHO line Lec13, rathybridoma cell line YB2/0, a cell line comprising a small interferingRNA specifically against the FUT8 gene, and a cell line coexpressingβ-1,4-N-acetylglucosaminyltransferase III and Golgi β-mannosidase II.Alternatively, the Fc-containing molecule may be expressed in anon-mammalian cell such as a plant cell, yeast, or prokaryotic cell,e.g., E. coli. Thus, in certain embodiments, a composition comprises anantibody having reduced fucosylation or lacking fucosylation altogether.

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No. 6,232,447 (LERK-6), U.S. Pat. No. 6,500,429(brain-derived neurotrophic factor), U.S. Pat. No. 6,184,359(epithelium-derived T-cell factor), U.S. Pat. No. 6,143,874(neurotrophic factor NNT-1), U.S. Patent Application Publication No.20110027287 (PROPROTEIN CONVERTASE SUBTILISIN KEXIN TYPE 9 (PCSK9)),U.S. Patent Application Publication No. 20110014201 (IL-18 RECEPTOR),and U.S. Patent Application Publication No. 20090155164 (C-FMS). Theabove patents and published patent applications are incorporated hereinby reference in their entirety for purposes of their disclosure ofvariable domain polypeptides, variable domain encoding nucleic acids,host cells, vectors, methods of making polypeptides encoding saidvariable domains, pharmaceutical compositions, and methods of treatingdiseases associated with the respective target of the variabledomain-containing antigen binding protein or antibody.

Antibodies and Fragments Thereof

The heterodimeric antibodies described herein, in some embodiments,comprise anti-sclerostin and anti-DKK1 antibodies and fragments thereofas described herein (e.g., a heterodimeric antibody comprising a heavyand light chain that mediates binding to sclerostin and a heavy andlight chain that mediates binding to DKK1). The term “antibody” refersto an intact antibody, or a binding fragment thereof. An antibody maycomprise a complete antibody (immunoglobulin) molecule (includingpolyclonal, monoclonal, chimeric, humanized, and/or human versionshaving full length heavy and/or light chains), or comprise an antigenbinding fragment thereof. Antibody fragments include F(ab′)₂, Fab, Fab′,Fv, Fc, and Fd fragments, and can be incorporated into single domainantibodies (e.g., nanobodies), single-chain antibodies, maxibodies,minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR andbis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology,23(9):1126-1136 (2005)). Antibody polypeptides, including fibronectinpolypeptide monobodies, also are disclosed in U.S. Pat. No. 6,703,199.Other antibody polypeptides are disclosed in U.S. Patent Publication No.20050238646.

An antibody fragment may be a synthetic or genetically engineeredprotein. For example, antibody fragments include isolated fragmentsconsisting of the light chain variable region, “Fv” fragments consistingof the variable regions of the heavy and light chains, and recombinantsingle chain polypeptide molecules in which light and heavy variableregions are connected by a peptide linker (scFv proteins).

Another form of an antibody fragment is a peptide comprising one or morecomplementarity determining regions (CDRs) of an antibody. As usedherein, the term “CDR” refers to the complementarity determining regionwithin antibody variable sequences. There are three CDRs in each of thevariable regions of the heavy chain and the light chain, which aredesignated CDR1, CDR2 and CDR3, for each of the variable regions. Theterm “CDR set” as used herein refers to a group of three CDRs that occurin a single variable region capable of binding the antigen. The exactboundaries of these CDRs have been defined differently according todifferent systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothiaet al., Nature 342:877-883 (1989)) found that certain sub-portionswithin Kabat CDRs adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3where the “L” and the “H” designates the light chain and the heavychains regions, respectively. These regions may be referred to asChothia CDRs, which have boundaries that overlap with Kabat CDRs. Otherboundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):73245 (1996)). Still other CDR boundary definitions may notstrictly follow one of the above systems, but will nonetheless overlapwith the Kabat CDRs, although they may be shortened or lengthened inlight of prediction or experimental findings that particular residues orgroups of residues or even entire CDRs do not significantly impactantigen binding. The methods used herein may utilize CDRs definedaccording to any of these systems, although preferred embodiments useKabat or Chothia defined CDRs.

CDRs (also termed “minimal recognition units” or “hypervariable region”)are obtained by, e.g., constructing polynucleotides that encode the CDRof interest. Such polynucleotides are prepared, for example, by usingthe polymerase chain reaction to synthesize the variable region usingmRNA of antibody-producing cells as a template (see, for example,Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106(1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,”in Monoclonal Antibodies Production, Engineering and ClinicalApplication, Ritter et al. (eds.), page 166, Cambridge University Press(1995); and Ward et al., “Genetic Manipulation and Expression ofAntibodies,” in Monoclonal Antibodies: Principles and Applications,Birch et al., (eds.), page 137, Wiley-Liss, Inc. (1995)).

The methods and antibody chains described herein are useful forgenerating heterodimeric antibodies. “Specifically binds” as used hereinmeans that the antigen binding protein preferentially binds the antigenover other proteins. In some embodiments “specifically binds” means theantigen binding protein has a higher affinity for the antigen than forother proteins. Antigen binding proteins that specifically bind anantigen may have a binding affinity for the antigen of less than orequal to 1×10⁻⁷ M, less than or equal to 2×10⁻⁷ M, less than or equal to3×10⁻⁷ M, less than or equal to 4×10⁻⁷ M, less than or equal to 5×10⁻⁷M, less than or equal to 6×10⁻⁷ M, less than or equal to 7×10⁻⁷ M, lessthan or equal to 8×10⁻⁷ M, less than or equal to 9×10⁻⁷ M, less than orequal to 1×10⁻⁸ M, less than or equal to 2×10⁻⁸ M, less than or equal to3×10⁻⁸ M, less than or equal to 4×10⁻⁸ M, less than or equal to 5×10⁻⁸M, less than or equal to 6×10⁻⁸ M, less than or equal to 7×10⁻⁸ M, lessthan or equal to 8×10⁻⁸ M, less than or equal to 9×10⁻⁸ M, less than orequal to 1×10⁻⁹ M, less than or equal to 2×10⁻⁹ M, less than or equal to3×10⁻⁹ M, less than or equal to 4×10⁻⁹ M, less than or equal to 5×10⁻⁹M, less than or equal to 6×10⁻⁹ M, less than or equal to 7×10⁻⁹ M, lessthan or equal to 8×10⁻⁹ M, less than or equal to 9×10⁻⁹ M, less than orequal to 1×10⁻¹⁰ M, less than or equal to 2×10⁻¹⁰ M, less than or equalto 3×10⁻¹⁰ M, less than or equal to 4×10⁻¹⁰ M, less than or equal to5×10⁻¹⁰ M, less than or equal to 6×10⁻¹⁰ M, less than or M, equal to7×10⁻¹⁰ M less than or equal to 8×10⁻¹⁰ M, less than or equal to 9×10⁻¹⁰M, less than or equal to 1×10⁻¹¹ M, less than or equal to 2×10⁻¹¹ M,less than or equal to 3×10⁻¹¹ M, less than or equal to 4×10⁻¹¹ M, lessthan or equal to 5×10⁻¹¹M, less than or equal to 6×10⁻¹¹ M, less than orequal to 7×10⁻¹¹ M, less than or equal to 8×10⁻¹¹ M, less than or equalto 9×10⁻¹¹ M, less than or equal to 1×10⁻¹² M, less than or equal to2×10⁻¹² M, less than or equal to 3×10⁻¹² M, less than or equal to4×10⁻¹² M, less than or equal to 5×10⁻¹² M, less than or equal to6×10⁻¹² M, less than or equal to 7×10⁻¹² M, less than or equal to8×10⁻¹² M, or less than or equal to 9×10⁻¹² M.

Anti-Sclerostin Antibodies

In some embodiments, the heterodimeric antibody described hereincomprises a sclerostin binding portion comprising an anti-sclerostinantibody. An “anti-sclerostin antibody” binds to sclerostin or portionsthereof to block or impair binding of human sclerostin to one or moreligands. Sclerostin, the product of the SOST gene, is absent insclerosteosis, a skeletal disease characterized by bone overgrowth andstrong dense bones (Brunkow et al., Am. J. Hum. Genet., 68:577-589(2001); Balemans et al., Hum. Mol. Genet., 10:537-543 (2001)). The aminoacid sequence of human sclerostin is reported by Brunkow et al. and isdisclosed in U.S. Patent Publication No. 20070110747 as SEQ ID NO: 1(which patent publication is incorporated in its entirety for itsdescription of sclerostin binding agents and Sequence Listing).Recombinant human sclerostin/SOST is commercially available from R&DSystems (Minneapolis, Minn., USA; 2006 Catalog #1406-ST-025).Additionally, recombinant mouse sclerostin/SOST is commerciallyavailable from R&D Systems (Minneapolis, Minn., USA; 2006 Catalog#1589-ST-025). Research grade sclerostin-binding monoclonal antibodiesare commercially available from R&D Systems (Minneapolis, Minn., USA;mouse monoclonal: 2006 Catalog #MAB1406; rat monoclonal: 2006 Catalog#MAB1589). U.S. Pat. Nos. 6,395,511 and 6,803,453, and U.S. PatentPublication Nos. 2004/0009535 and 2005/0106683 refer to anti-sclerostinantibodies generally. Examples of sclerostin binding agents suitable foruse in the context of the invention also are described in U.S. PatentPublication Nos. 2007/0110747 and 2007/0072797, which are herebyincorporated by reference. Additional information regarding materialsand methods for generating sclerostin binding agents can be found inU.S. Patent Publication No. 20040158045 (hereby incorporated byreference).

Anti-sclerostin antibodies or fragments thereof may bind to sclerostinof SEQ ID NO: 1, or a naturally occurring variant thereof, with anaffinity (Kd) of less than or equal to 1×10⁻⁷ M, less than or equal to1×10⁻⁸M, less than or equal to 1×10⁻⁹ M, less than or equal to 1×10⁻¹⁰M, less than or equal to 1×10⁻¹¹ M, or less than or equal to 1×10⁻¹² M.Affinity is determined using a variety of techniques, an example ofwhich is an affinity ELISA assay. In various embodiments, affinity isdetermined by a BIAcore assay. In various embodiments, affinity isdetermined by a kinetic method. In various embodiments, affinity isdetermined by an equilibrium/solution method. U.S. Patent PublicationNo. 2007/0110747 contains additional description of affinity assayssuitable for determining the affinity (Kd) of an antibody forsclerostin.

In some or any embodiments, the anti-sclerostin antibody or antibodyfragment binds to a sclerostin polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 1 and binds a region of sclerostincomprising the sequence of SEQ ID NO: 6 (CGPARLLPNAIGRGKWWRPSGPDFRC;corresponding to amino acids 86-111 of SEQ ID NO: 1). This region isalso referred to herein as the “loop 2” region of sclerostin. Regions ofsclerostin outside of the loop 2 region are defined herein as “non-loop2 regions.” Alternatively or in addition, the anti-sclerostin antibodybinds to a sclerostin polypeptide comprising amino acids 57-146 of SEQID NO: 1. Alternatively or in addition, the anti-sclerostin antibodybinds to a sclerostin polypeptide comprising amino acids 89-103 of SEQID NO: 1 and/or amino acids 137-151 of SEQ ID NO: 1. Alternatively or inaddition, the anti-sclerostin antibody binds to a sclerostin polypeptidecomprising the amino acid sequence set forth in SEQ ID NO: 1 and bindsthe sequence of at least one of SEQ ID NO: 2 (DVSEYSCRELHFTR;corresponding to amino acids 51-64 of SEQ ID NO: 1), SEQ ID NO: 3(SAKPVTELVCSGQCGPAR; corresponding to amino acids 73-90 of SEQ ID NO:1), SEQ ID NO: 4 (WWRPSGPDFRCIPDRYR; corresponding to amino acids101-117 of SEQ ID NO: 1), SEQ ID NO: 5 (LVASCKCKRLTR; corresponding toamino acids 138-149 of SEQ ID NO: 1), SEQ ID NO: 70 (SAKPVTELVCSGQC;corresponding to amino acids 73-86 of SEQ ID NO: 1), SEQ ID NO: 71(LVASCKC; corresponding to amino acids 138-144 of SEQ ID NO: 1), SEQ IDNO: 72 (C1RELHFTR; corresponding to amino acids 57-64 of SEQ ID NO: 1),or SEQ ID NO: 73 (CIPDRYR; corresponding to amino acids 111-117 of SEQID NO: 1) within SEQ ID NO: 1. For example, in one aspect, theanti-sclerostin antibody binds a subregion of sclerostin of SEQ ID NO: 1comprising SEQ ID NOs: 2-5 (and/or SEQ ID NOs: 70-73), optionally in itsnative three-dimensional conformation. Optionally, the anti-sclerostinantibody binds a peptide consisting of one or more of SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 70, SEQID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73 (e.g., a peptide consistingof SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 or apeptide consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, andSEQ ID NO: 73).

In some or any embodiments, the anti-sclerostin antibody binds to asclerostin polypeptide comprising amino acids 89-103 and 137-151 of SEQID NO: 1.

In some or any embodiments, the anti-sclerostin antibody binds to asclerostin polypeptide having the amino acid sequences of SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, wherein SEQ ID NO:2 and 4 arejoined by a disulfide bond at amino acid positions 57 and 111 withreference to SEQ ID NO:1, and SEQ ID NO:3 and 5 are joined by at leastone of (a) a disulfide bond at amino acid positions 82 and 142 withreference to SEQ ID NO:1, and (b) a disulfide bond at amino acidpositions 86 and 144 with reference to SEQ ID NO:1; the polypeptide mayretain the tertiary structure of the corresponding polypeptide region ofhuman sclerostin of SEQ ID NO:1. Alternatively or in addition, theanti-sclerostin antibody binds a polypeptide having the amino acidsequences of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO:73, wherein SEQ ID NO: 72 and 73 are joined by a disulfide bond at aminoacid positions 57 and 111 with reference to SEQ ID NO: 1, and SEQ ID NO:70 and 71 are joined by at least one of (a) a disulfide bond at aminoacid positions 82 and 142 with reference to SEQ ID NO: 1, and (b) adisulfide bond at amino acid positions 86 and 144 with reference to SEQID NO: 1.

Optionally, the anti-sclerostin antibody binds a peptide consistingessentially of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4 and SEQ ID NO: 5, wherein SEQ ID NO: 2 and 4 are joined bya disulfide bond at amino acid positions 57 and 111 with reference toSEQ ID NO: 1, and SEQ ID NO: 3 and 5 are joined by at least one of (a) adisulfide bond at amino acid positions 82 and 142 with reference to SEQID NO: 1, and (b) a disulfide bond at amino acid positions 86 and 144with reference to SEQ ID NO: 1.

Optionally, the anti-sclerostin antibody binds to a polypeptideconsisting essentially of a multiply truncated human sclerostin proteinof SEQ ID NO: 1, wherein (a) amino acids 1-50, 65-72, 91-100, 118-137,and 150-190 of SEQ ID NO: 1 are absent from said polypeptide or (b)amino acids 1-56, 65-72, 87-110, 118-137, and 145-190 of SEQ ID NO: 1are absent from said polypeptide.

In some or any embodiments, the anti-sclerostin antibody binds to apolypeptide having the amino acid sequences of SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO: 72 and SEQ ID NO: 73, wherein SEQ ID NO: 72 and 73 arejoined by a disulfide bond at amino acid positions 57 and 111 withreference to SEQ ID NO: 1, and SEQ ID NO: 70 and 71 are joined by atleast one of (a) a disulfide bond at amino acid positions 82 and 142with reference to SEQ ID NO: 1, and (b) a disulfide bond at amino acidpositions 86 and 144 with reference to SEQ ID NO: 1.

In some or any embodiments, the sclerostin polypeptide retains thetertiary structure of the corresponding polypeptide region of humansclerostin of SEQ ID NO: 1.

In some or any embodiments, the anti-sclerostin antibody binds to (i) aportion of human sclerostin comprising amino acids 51-64, 73-90,101-117, and 138-149 of SEQ ID NO: 1, wherein said portion has at leastone, at least two or all three of: (a) a disulfide bond between aminoacids 57 and 111; (b) a disulfide bond between amino acids 82 and 142;and (c) a disulfide bond between amino acids 86 and 144; or (ii) aportion of human sclerostin comprising amino acids 57-64, 73-86,111-117, and 138-144 of SEQ ID NO: 1, wherein said portion has at leastone, at least two, or all three of: (a) a disulfide bond between aminoacids 57 and 111; (b) a disulfide bond between amino acids 82 and 142;and (c) a disulfide bond between amino acids 86 and 144.

In some or any embodiments, the anti-sclerostin antibody also binds toan epitope of SEQ ID NO: 6.

Anti-sclerostin antibodies for use in generating the heterodimericantibodies described herein preferably modulate sclerostin function inthe cell-based assay described in U.S. Patent Publication No.2007/0110747 and/or the in vivo assay described in U.S. PatentPublication No. 20070110747 and/or bind to one or more of the epitopesdescribed in U.S. Patent Publication No. 2007/0110747 and/or cross-blockthe binding of one of the antibodies described in U.S. PatentPublication No. 2007/0110747 and/or are cross-blocked from bindingsclerostin by one of the antibodies described in U.S. Patent PublicationNo. 2007/0110747 (incorporated by reference in its entirety and for itsdescription of assays for characterizing an anti-sclerostin antibody).

In various aspects, the anti-sclerostin antibody is also capable ofneutralizing human sclerostin in a MC3T3 cell-based mineralization assaywhen there is less than a 6-fold excess of moles of sclerostin bindingsites per well as compared to the number of moles of sclerostin perwell. Mineralization by osteoblast-lineage cells in culture, eitherprimary cells or cell lines, is used as an in vitro model of boneformation. An exemplary cell-based mineralization assay is described inU.S. Patent Publication No. 20070110747 at, e.g., Example 8 (herebyincorporated by reference). MC3T3-E1 cells (Sudo et al., J. Cell Biol.,96:191-198 (1983)) and subclones of the original cell line can formmineral in culture upon growth in the presence of differentiatingagents. Such subclones include MC3T3-E1-BF (Smith et al., J. Biol.Chem., 275:19992-20001 (2000)). For both the MC3T3-E1-BF subclone aswell as the original MC3T3-E1 cells, sclerostin can inhibit one or moreof the sequence of events leading up to and including mineral deposition(i.e., sclerostin inhibits mineralization). Anti-sclerostin antibodiesthat are able to neutralize sclerostin's inhibitory activity allow formineralization of the culture in the presence of sclerostin such thatthere is a statistically significant increase in, e.g., deposition ofcalcium phosphate (measured as calcium) as compared to the amount ofcalcium measured in the sclerostin-only (i.e., no antibody) treatmentgroup.

When running the assay with the goal of determining whether a particularanti-sclerostin antibody (or other sclerostin inhibitor) can neutralizesclerostin, the amount of sclerostin used in the assay desirably is theminimum amount of sclerostin that causes at least a 70%, statisticallysignificant, reduction in deposition of calcium phosphate (measured ascalcium) in the sclerostin-only group, as compared to the amount ofcalcium measured in the no sclerostin group. An anti-sclerostinneutralizing antibody is defined as one that causes a statisticallysignificant increase in deposition of calcium phosphate (measured ascalcium) as compared to the amount of calcium measured in thesclerostin-only (i.e., no antibody) treatment group. To determinewhether an anti-sclerostin antibody is neutralizing or not, the amountof anti-sclerostin antibody used in the assay needs to be such thatthere is an excess of moles of sclerostin binding sites per well ascompared to the number of moles of sclerostin per well. Depending on thepotency of the antibody, the fold excess that may be required can be 24,18, 12, 6, 3, or 1.5, and one of skill is familiar with the routinepractice of testing more than one concentration of binding agent(antibody). For example, a very potent anti-sclerostin neutralizingantibody will neutralize sclerostin when there is less than a 6-foldexcess of moles of sclerostin binding sites per well as compared to thenumber of moles of sclerostin per well. A less potent anti-sclerostinneutralizing antibody will neutralize sclerostin only at a 12, 18 or 24fold excess.

The anti-sclerostin antibody optionally has an IC50 of 100 nM or less,or 75 nM or less, or 50 nM or less, or 25 nM or less for neutralizinghuman sclerostin in a cell-based assay, such as a bone specific alkalinephosphatase assay, e.g., the bone specific alkaline phosphatase assaydescribed in International Patent Publication No. WO 2008/115732 andU.S. Pat. No. 7,744,874 (incorporated herein by reference in itsentirety for its description of cell-based assays and anti-sclerostinantibodies). The bone specific alkaline phosphatase assay is predicatedon the ability of sclerostin to decrease BMP-4 and Wnt3a-stimulatedalkaline phosphatase levels in the multipotential murine cell line,C2C12. According to WO 2008/115732, a neutralizing anti-sclerostinantibody mediates a dose-dependent increase of alkaline phosphataseactivity in this assay.

Alternatively or in addition, the anti-sclerostin antibody has an IC50of 100 nM or less (e.g., 75 nM or less, or 50 nM or less) forneutralizing human sclerostin in a cell-based Wnt signaling assay inHEK293 cell lines, such as the Wnt assay involving Wnt1-mediatedinduction of STF reporter gene described in e.g., International PatentPublication No. WO 2009/047356 (incorporated by reference for itsdiscussion of anti-sclerostin antibodies and cell-based assays).Alternatively or in addition, the anti-sclerostin antibody has an IC50of 500 nM or less (e.g., 250 nM or less, 150 nM or less, 100 nM or less,or 50 nM or less) for neutralizing human sclerostin in a BMP2-inducedmineralization assay in MC3T3 cells, such as the mineralization assaydescribed in e.g., International Patent Publication No. WO 2009/047356.

Examples of anti-sclerostin antibodies suitable for use in the contextof the invention are described in U.S. Patent Publication Nos.2007/0110747 and 2007/0072797, which are hereby incorporated byreference. In some embodiments, the anti-sclerostin antibodycross-blocks the binding of at least one of antibodies Ab-A, Ab-B, Ab-C,Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10,Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20,Ab-21, Ab-22, Ab-23, and Ab-24 (all of which are described in U.S.Patent Publication No. 20070110747) to sclerostin. Alternatively or inaddition, the anti-sclerostin antibody is cross-blocked from binding tosclerostin by at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1,Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12,Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22,Ab-23, and Ab-24 (all of which are described in U.S. Patent PublicationNo. 20070110747). The terms “cross-block,” “cross-blocked,” and“cross-blocking” are used interchangeably herein to mean the ability ofan antibody to interfere with the binding of other antibodies tosclerostin. The extent to which an antibody is able to interfere withthe binding of another to sclerostin, and therefore whether it can besaid to cross-block, can be determined using competition binding assays.In some aspects of the invention, a cross-blocking antibody or fragmentthereof reduces sclerostin binding of a reference antibody between about40% and about 100%, such as about 60% and about 100%, specificallybetween 70% and 100%, and more specifically between 80% and 100%. Aparticularly suitable quantitative assay for detecting cross-blockinguses a Biacore machine which measures the extent of interactions usingsurface plasmon resonance technology. Another suitable quantitativecross-blocking assay uses an ELISA-based approach to measure competitionbetween antibodies in terms of their binding to sclerostin.

In some embodiments, the anti-sclerostin antibody cross-blocks thebinding of an immunoglobulin comprising full length heavy and lightchains to sclerostin of SEQ ID NO: 1 and/or is cross-blocked frombinding to sclerostin of SEQ ID NO: 1 by an immunoglobulin comprisingfull length heavy and light chains, wherein the immunoglobulincomprising full length heavy and light chains comprise CDR sequencesdisclosed herein, such as one of the following three sets of CDRsequences: a) CDR-L1 of SEQ ID NO: 284, CDR-L2 of SEQ ID NO: 285, CDR-L3of SEQ ID NO: 286, CDR-H1 of SEQ ID NO: 296, CDR-H2 of SEQ ID NO: 297,and CDR-H3 of SEQ ID NO: 298; b) CDR-L1 of SEQ ID NO: 48, CDR-L2 of SEQID NO: 49, CDR-L3 of SEQ ID NO: 50, CDR-H1 of SEQ ID NO: 45, CDR-H2 ofSEQ ID NO: 46, and CDR-H3 of SEQ ID NO: 47; or c) CDR-L1 of SEQ ID NO:42, CDR-L2 of SEQ ID NO: 43, CDR-L3 of SEQ ID NO: 44, CDR-H1 of SEQ IDNO: 39, CDR-H2 of SEQ ID NO: 40, and CDR-H3 of SEQ ID NO: 41.Alternatively, or in addition, the anti-sclerostin antibody cross-blocksthe binding of immunoglobulin comprising full length heavy and lightchains to sclerostin of SEQ ID NO: 1 and/or is cross-blocked frombinding to sclerostin of SEQ ID NO: 1 by an immunoglobulin comprisingfull length heavy and light chains, wherein the immunoglobulincomprising full length heavy and light chains comprise the followingCDRs: CDR-H1 of SEQ ID NO: 245, CDR-H2 of SEQ ID NO: 246, CDR-H3 of SEQID NO: 247, CDR-L1 of SEQ ID NO: 78, CDR-L2 of SEQ ID NO: 79 and CDR-L3of SEQ ID NO: 80.

Alternatively, or in addition, the anti-sclerostin antibody cross-blocksthe binding of immunoglobulin comprising full length heavy and lightchains to sclerostin of SEQ ID NO: 1 and/or is cross-blocked frombinding to sclerostin of SEQ ID NO: 1 by an immunoglobulin comprisingfull length heavy and light chains, wherein the immunoglobulincomprising full length heavy and light chains comprise the followingCDRs: CDR-H1 of SEQ ID NO: 269, CDR-H2 of SEQ ID NO: 270, CDR-H3 of SEQID NO: 271, CDR-L1 of SEQ ID NO: 239, CDR-L2 of SEQ ID NO: 240 andCDR-L3 of SEQ ID NO: 241.

Examples of suitable anti-sclerostin antibodies and fragments thereofinclude antibodies and antibody fragments having one or more of CDR-H1,CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 specifically disclosed hereinand disclosed in U.S. Patent Publication No. 2007/0110747. At least oneof the regions of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 mayhave at least one amino acid substitution, provided that the antibodyretains the binding specificity of the non-substituted CDR. Exemplarythe anti-sclerostin antibodies include, but are not limited to, Ab-A,Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9,Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19,Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24 of U.S. Patent Publication No.2007/0110747. Other exemplary anti-sclerostin antibodies include, butare not limited to, 27H6, 19D11 and 20C3.

In addition, the anti-sclerostin antibody can comprise at least one CDRsequence having at least 75% identity (e.g., 100% identity) to a CDRselected from SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 78, 79, 80, 81, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298, 351, 352, 353, 358, 359,and 360 provided in the Sequence Listing and disclosed in U.S. PatentPublication No. 20070110747. In addition, the anti-sclerostin antibodycan comprise at least one CDR sequence having at least 75% identity(e.g., 100% identity) to a CDR selected from SEQ ID NOs: 417-422,425-430 and 433-438 provided in the Sequence Listing. Preferably, theanti-sclerostin antibody comprises at least one CDR sequence having atleast 75% identity to a CDR selected from SEQ ID NOs: 245, 246, 247, 78,79, 80, 269, 270, 271, 239, 240, and 241, all of which is provided inthe Sequence Listing and described in U.S. Patent Publication No.20070110747. As described in U.S. Patent Publication No. 2007/0110747,the anti-sclerostin antibody can comprise: a) CDR sequences of SEQ IDNOs:54, 55, and 56 and CDR sequences of SEQ ID NOs:51, 52, and 53; b)CDR sequences of SEQ ID NOs:60, 61, and 62 and CDR sequences of SEQ IDNOs:57, 58, and 59; c) CDR sequences of SEQ ID NOs:48, 49, and 50 andCDR sequences of SEQ ID NOs:45, 46, and 47; d) CDR sequences of SEQ IDNOs:42, 43, and 44 and CDR sequences of SEQ ID NOs:39, 40, and 41; e)CDR sequences of SEQ ID NOs:275, 276, and 277 and CDR sequences of SEQID NOs:287, 288, and 289; f) CDR sequences of SEQ ID NOs:278, 279, and280 and CDR sequences of SEQ ID NOs:290, 291, and 292; g) CDR sequencesof SEQ ID NOs:78, 79, and 80 and CDR sequences of SEQ ID NOs: 245, 246,and 247; h) CDR sequences of SEQ ID NOs:81, 99, and 100 and CDRsequences of SEQ ID NOs:248, 249, and 250; i) CDR sequences of SEQ IDNOs:101, 102, and 103 and CDR sequences of SEQ ID NOs:251, 252, and 253;j) CDR sequences of SEQ ID NOs:104, 105, and 106 and CDR sequences ofSEQ ID NOs:254, 255, and 256; k) CDR sequences of SEQ ID NOs:107, 108,and 109 and CDR sequences of SEQ ID NOs:257, 258, and 259; l) CDRsequences of SEQ ID NOs:110, 111, and 112 and CDR sequences of SEQ IDNOs:260, 261, and 262; m) CDR sequences of SEQ ID NOs:281, 282, and 283and CDR sequences of SEQ ID NOs:293, 294, and 295; n) CDR sequences ofSEQ ID NOs:113, 114, and 115 and CDR sequences of SEQ ID NOs:263, 264,and 265; o) CDR sequences of SEQ ID NOs:284, 285, and 286 and CDRsequences of SEQ ID NOs:296, 297, and 298; p) CDR sequences of SEQ IDNOs:116, 237, and 238 and CDR sequences of SEQ ID NOs:266, 267, and 268;q) CDR sequences of SEQ ID NOs:239, 240, and 241 and CDR sequences ofSEQ ID NOs:269, 270, and 271) CDR sequences of SEQ ID NOs:242, 243, and244 and CDR sequences of SEQ ID NOs:272, 273, and 274; or s) CDRsequences of SEQ ID NOs:351, 352, and 353 and CDR sequences of SEQ IDNOs:358, 359, and 360.

The anti-sclerostin antibody can comprise at least one CDR sequencehaving at least 75% identity (e.g., 100% identical) to a CDR selectedfrom CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 wherein CDR-H1has the sequence given in SEQ ID NO: 245, CDR-H2 has the sequence givenin SEQ ID NO: 246, CDR-H3 has the sequence given in SEQ ID NO: 247,CDR-L1 has the sequence given in SEQ ID NO: 78, CDR-L2 has the sequencegiven in SEQ ID NO: 79 and CDR-L3 has the sequence given in SEQ ID NO:80, all of which is provided in the Sequence Listing and described inU.S. Patent Publication No. 20070110747. The anti-sclerostin antibody,in various aspects, comprises two of the CDRs or six of the CDRs.Optionally, the anti-sclerostin antibody comprises all or part of aheavy chain (e.g., two heavy chains) comprising SEQ ID NO: 378 and allor part of a light chain (e.g., two light chains) comprising SEQ ID NO376.

The anti-sclerostin antibody can comprise at least one CDR sequencehaving at least 75% identity (e.g., 100% identical) to a CDR selectedfrom CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 wherein CDR-H1has the sequence given in SEQ ID NO: 269, CDR-H2 has the sequence givenin SEQ ID NO: 270, CDR-H3 has the sequence given in SEQ ID NO: 271,CDR-L1 has the sequence given in SEQ ID NO: 239, CDR-L2 has the sequencegiven in SEQ ID NO: 240 and CDR-L3 has the sequence given in SEQ ID NO241, all of which is provided in the Sequence Listing and described inU.S. Patent Publication No. 20070110747. The anti-sclerostin antibody,in various aspects, comprises at least two of the CDRs or six of theCDRs. Optionally, the anti-sclerostin antibody comprises all or part ofa heavy chain (e.g., two heavy chains) comprising SEQ ID NO: 366 and allor part of a light chain (e.g., two light chains) comprising SEQ ID NO364.

Alternatively, the anti-sclerostin antibody can have a heavy chaincomprising CDR's H1, H2, and H3 and comprising a polypeptide having thesequence provided in SEQ ID NO: 137, 145, or 392 or a variant thereof inwhich the CDRs are at least 75% identical (e.g., 100% identical) to SEQID NO: 245, 246, and 247, respectively, and a light chain comprisingCDR's L1, L2 and L3 and comprising a polypeptide having the sequenceprovided in SEQ ID NO: 133 or 141 or a variant thereof in which the CDRsare at least 75% identical (e.g., 100% identical) to SEQ ID NO: 78, 79,and 80, respectively (as described in U.S. Patent Publication No.2007/0110747).

The anti-sclerostin antibody may have a heavy chain comprising CDR's H1,H2, and H3 and comprising a polypeptide having the sequence provided inSEQ ID NO: 335, 331, 345, or 396 or a variant of any of the foregoing inwhich the CDRs are at least 75% (e.g., 100% identical) identical to SEQID NO: 269, 270, and 271, respectively, and a light chain comprisingCDR's L1, L2, and L3 and comprising a polypeptide having the sequenceprovided in SEQ ID NO: 334 or 341 or a variant of any of the foregoingin which the CDRs are at least 75% identical (e.g., 100% identical) toSEQ ID NO: 239, 240, and 241, respectively (as described in U.S. PatentPublication No. 20070110747). All combinations of the heavy and lightchain sequences are contemplated (e.g., heavy chains comprising SEQ IDNO: 335 and light chains comprising SEQ ID NO: 334; heavy chainscomprising SEQ ID NO: 331 and light chains comprising SEQ ID NO: 334 or341; and heavy chains comprising SEQ ID NO: 345 or 396 and light chainscomprising SEQ ID NO: 341).

Alternatively, the anti-sclerostin antibody has a heavy chain comprisinga polypeptide having the sequence provided in SEQ ID NO:137, and a lightchain comprising a polypeptide having the sequence provided in SEQ IDNO:133; a heavy chain comprising a polypeptide having the sequenceprovided in SEQ ID NO:145 or 392, and a light chain comprising apolypeptide having the sequence provided in SEQ ID NO: 141; a heavychain comprising a polypeptide having the sequence provided in SEQ IDNO:335, and a light chain comprising a polypeptide having the sequenceprovided in SEQ ID NO:334; a heavy chain comprising a polypeptide havingthe sequence provided in SEQ ID NO:331, and a light chain comprising apolypeptide having the sequence provided in SEQ ID NO:341; or a heavychain comprising a polypeptide having the sequence provided in SEQ IDNO:345 or 396, and a light chain comprising a polypeptide having thesequence provided in SEQ ID NO:341 (as described in U.S. PatentPublication No. 2007/0110747). Alternatively, the anti-sclerostinantibody cross-blocks (or is cross-blocked by) any of the aforementionedantibodies to sclerostin.

Examples of anti-sclerostin antibodies also include, but are not limitedto, the anti-sclerostin antibodies disclosed in International PatentPublication Nos. WO 2008/092894, WO 2008/115732, WO 2009/056634, WO2009/047356, WO 2010/100200, WO 2010/100179, WO 2010/115932, and WO2010/130830 (each of which is incorporated by reference herein in itsentirety), such as an anti-sclerostin antibody comprising CDRs of SEQ IDNOs: 20-25 of International Patent Publication No. WO 2008/115732 (SEQID NOs: 416-421 herein), an anti-sclerostin antibody comprising CDRs ofSEQ ID NOs: 26-31 of International Patent Publication No. WO 2008/115732(SEQ ID NOs: 422-427 herein), an anti-sclerostin antibody comprisingCDRs of SEQ ID NOs: 32-37 of International Patent Publication No. WO2008/115732 (SEQ ID NOs: 428-433 herein), an anti-sclerostin antibodycomprising CDRs of SEQ ID NOs: 4, 15, 26, 37, 48, and 59 ofInternational Patent Publication No. WO 2009/047356 (SEQ ID NOs: 443,454, 465, 476, 487 and 498, respectively, herein), or an anti-sclerostinantibody comprising the amino acid sequence of at least one of SEQ IDNOs: 135-143, 153-161, or 171-179 of International Patent PublicationNo. WO 2010/130830 (SEQ ID NOs: 745-753, 763-771, 781-789, respectively,herein).

Anti-DKK1 Antibodies

In some embodiments, the heterodimeric antibody described hereincomprises a DKK1 binding portion comprising an anti-DKK1 antibody. An“anti-DKK1 antibody” binds to DKK1 or portions thereof to block orimpair binding of human DKK1 to one or more ligands. Human DKK1polynucleotide and amino acid sequences are set forth in SEQ ID NOs: 810and 811, respectively. Polynucleotide and amino acid sequences for mouseand rat DKK1 are set forth in SEQ ID NOs: 812 and 813 (mouse) and SEQ IDNOs: 814 and 815 (rat). Examples of anti-DKK1 antibodies suitable foruse in the context of the invention are described in InternationalPublication No. WO 2012/118903, the disclosure of which is incorporatedherein by reference. In some embodiments, the anti-DKK1 antibodycross-blocks or competes with the binding of at least one of Antibodies11H10Hu, 11H10Rat, 2.4.1, 2.20.1, 2.37.1, 2.40.1, 2.41.1, 2.47.1,5.17.1, 5.23.1, 5.25.1, 5.31.1, 5.32.1, 5.40.1, 5.65.1, 5.76.1, 5.77.1,5.78.1, 5.80.1, 5.85.1, 6.37.5, 6.116.6, 6.139.5 and 6.147.4 (all ofwhich are described in International Publication No. WO 2012/118903) toDKK1. Alternatively, or in addition, the anti-DKK1 antibody iscross-blocked from binding to DKK1 by at least one of antibodies11H10Hu, 11H10Rat, 2.4.1, 2.20.1, 2.37.1, 2.40.1, 2.41.1, 2.47.1,5.17.1, 5.23.1, 5.25.1, 5.31.1, 5.32.1, 5.40.1, 5.65.1, 5.76.1, 5.77.1,5.78.1, 5.80.1, 5.85.1, 6.37.5, 6.116.6, 6.139.5 and 6.147.4. The terms“cross-block,” “cross-blocked,” and “cross-blocking” are usedinterchangeably herein to mean the ability of an antibody to interferewith the binding of other antibodies to DKK1. The extent to which anantibody is able to interfere with the binding of another to DKK1, andtherefore whether it can be said to cross-block, can be determined usingcompetition binding assays. In some aspects, a cross-blocking antibodyor fragment thereof reduces DKK1 binding of a reference antibody betweenabout 40% and about 100%, such as about 60% and about 100%, or between70% and 100%, or between 80% and 100%. A particularly suitablequantitative assay for detecting cross-blocking uses a Biacore machinewhich measures the extent of interactions using surface plasmonresonance technology. Another suitable quantitative cross-blocking assayuses an ELISA-based approach to measure competition between antibodiesin terms of their binding to DKK1.

In some embodiments, the anti-DKK1 antibody cross-blocks the binding ofan immunoglobulin comprising full length heavy and light chains to DKK1of SEQ ID NO: 811 and/or is cross-blocked from binding to DKK1 of SEQ IDNO: 811 by an immunoglobulin comprising full length heavy and lightchains, wherein the immunoglobulin comprising full length heavy andlight chains comprises CDR sequences disclosed herein, such as one ofthe following three sets of CDR sequences: SEQ ID NOs: 820-822, 828-830,836-838, 844-846, 852-854, 860-862, 868-869, 876-878, 884-886, 892-894,900-902, 908-910, 916-918, 925-927, 932-934, 940-942, 948-950, 956-958,964-966, 972-974, 980-982, 988-990, 996-998, 1004-1006, 823-825,831-833, 839-841, 847-849, 855-857, 863-865, 871-873, 879-881, 887-889,897-897, 903-905, 911-913, 919-921, 927-929, 935-937, 943-945, 951-953,959-961, 967-969, 975-977, 983-985, 991-993, 999-1001 and 1007-1009.

Examples of suitable anti-DKK1 antibodies and fragments thereof includeantibodies and antibody fragments having one or more of CDR-H1, CDR-H2,CDR-H3, CDR-L1, CDR-L2 and CDR-L3 specifically disclosed herein anddisclosed in International Publication No. WO 2012/118903, which isincorporated herein by reference in its entirety. In some embodiments,at least one of the regions of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2,and CDR-L3 may have at least one amino acid substitution, provided thatthe antibody retains the binding specificity of the non-substituted CDR.Exemplary anti-DKK1 antibodies include, but are not limited to,Antibodies 11H10Hu, 11H10Rat, 2.4.1, 2.20.1, 2.37.1, 2.40.1, 2.41.1,2.47.1, 5.17.1, 5.23.1, 5.25.1, 5.31.1, 5.32.1, 5.40.1, 5.65.1, 5.76.1,5.77.1, 5.78.1, 5.80.1, 5.85.1, 6.37.5, 6.116.6, 6.139.5 and 6.147.4(all of which are described in International Publication No. WO2012/118903).

In some embodiments, the anti-DKK1 antibody comprises at least one CDRhaving at least 75% identity (e.g., at least 80%, or at least 85%, or atleast 90%, or at least 95%, or at least 100% identity) to a CDR selectedfrom the group consisting of 820-822 (CDRL1-L3 of Ab 11H10Hu), 828-830(CDRL1-CDRL3 of Ab 11H10Rat), 836-838 (CDRL1-CDRL3 of Ab 2.4.1), 844-846(CDRL1-CDRL3 of Ab 2.20.1), 852-854 (CDRL1-CDRL3 of Ab 2.37.1), 860-862(CDRL1-CDRL3 of Ab 2.40.1), 868-869 (CDRL1-CDRL3 of Ab 2.41.1), 876-878(CDRL1-CDRL3 of Ab2.47.1), 884-886 (CDRL1-CDRL3 of Ab 5.17.1), 892-894(CDRL1-CDRL3 of Ab 5.23.1), 900-902 (CDRL1-CDRL3 of Ab 5.25.1), 908-910(CDRL1-CDRL3 of Ab5.31.1), 916-918 (CDRL1-CDRL3 of Ab 5.32.1), 925-927(CDRL1-CDRL3 of Ab 5.40.1), 932-934 (CDRL1-CDRL3 of Ab 5.65.1), 940-942(CDRL1-CDRL3 of Ab 5.76.1), 948-950 (CDRL1-CDRL3 of Ab5.77.1), 956-958(CDRL1-CDRL3 of Ab 5.78.1), 964-966 (CDRL1-CDRL3 of Ab 5.80.1), 972-974(CDRL1-CDRL3 of Ab 5.85.1), 980-982 (CDRL1-CDRL3 of Ab 6.37.5), 988-990(CDRL1-CDRL3 of Ab 6.116.6), 996-998 (CDRL1-CDRL3 of Ab 6.139.5),1004-1006 (CDRL1-CDRL3 of Ab 6.147.4), 823-825 (CDRH1-CDRH3 of Ab11H10Hu), 831-833 (CDRH1-CDRH3 of Ab 11H10Rat), 839-841 (CDRH1-CDRH3 ofAb 2.4.1), 847-849 (CDRH1-CDRH3 of Ab 2.20.1), 855-857 (CDRH1-CDRH3 ofAb 2.37.1), 863-865 (CDRH1-CDRH3 of Ab 2.40.1), 871-873 (CDRH1-CDRH3 ofAb 2.41.1), 879-881 (CDRH1-CDRH3 of Ab 2.47.1), 887-889 (CDRH1-CDRH3 ofAb 5.17.1), 895-897 (CDRH1-CDRH3 of Ab 5.23.1), 903-905 (CDRH1-CDRH3 ofAb 5.25.1), 911-913 (CDRH1-CDRH3 of Ab 531.1), 919-921 (CDRH1-CDRH3 ofAb 5.32.1), 927-929 (CDRH1-CDRH3 of Ab 5.40.1), 935-937 (CDRH1-CDRH3 ofAb 5.65.1), 943-945 (CDRH1-CDRH3 of Ab 5.76.1), 951-953 (CDRH1-CDRH3 ofAb 5.77.1), 959-961 (CDRH1-CDRH3 of Ab 5.78.1), 967-969 (CDRH1-CDRH3 ofAb 5.80.1), 975-977 (CDRH1-CDRH3 of Ab5.85.1), 983-985 (CDRH1-CDRH3 ofAb 6.37.5), 991-993 (CDRH1-CDRH3 of Ab 6.116.6), 999-1001 (CDRH1-CDRH3of Ab 6.139.5) and 1007-1009 (CDRH1-CDRH3 of Ab 6.147.4).

The anti-DKK1 antibody comprises, in some embodiments, having a heavychain variable domain amino acid sequence having at least 75% identity(e.g., at least 80%, or at least 85%, or at least 90%, or at least 95%,or at least 100% identity) to an anti-DKK1 heavy chain variable domainamino acid sequence selected from the group consisting of SEQ ID NOs:819, 827, 835, 843, 851, 859, 867, 875, 883, 891, 899, 907, 915, 923,931, 939, 947, 955, 963, 971, 979, 987, 995 and 1003. In someembodiments, the anti-DKK1 antibody comprising a light chain variabledomain amino acid sequence having at least 75% identity (e.g., at least80%, or at least 85%, or at least 90%, or at least 95%, or at least 100%identity) to an anti-DKK1 light chain variable domain amino acidsequence selected from the group consisting of SEQ ID NOs: 818, 826,834, 842, 850, 866, 874, 882, 890, 898, 906, 814, 822, 830, 938, 946,954, 962, 970, 978, 988, 994 and 1002.

The DKK1 binding component(s) of the heterodimeric antibody comprises,in some embodiments, one, two, three, four, five or six of the CDRs setforth in SEQ ID NOs: of 820-822 (CDRL1-L3 of Ab 11H10Hu), 828-830(CDRL1-CDRL3 of Ab 11H10Rat), 836-838 (CDRL1-CDRL3 of Ab 2.4.1), 844-846(CDRL1-CDRL3 of Ab 2.20.1), 852-854 (CDRL1-CDRL3 of Ab 2.37.1), 860-862(CDRL1-CDRL3 of Ab 2.40.1), 868-869 (CDRL1-CDRL3 of Ab 2.41.1), 876-878(CDRL1-CDRL3 of Ab2.47.1), 884-886 (CDRL1-CDRL3 of Ab 5.17.1), 892-894(CDRL1-CDRL3 of Ab 5.23.1), 900-902 (CDRL1-CDRL3 of Ab 5.25.1), 908-910(CDRL1-CDRL3 of Ab5.31.1), 916-918 (CDRL1-CDRL3 of Ab 5.32.1), 925-927(CDRL1-CDRL3 of Ab 5.40.1), 932-934 (CDRL1-CDRL3 of Ab 5.65.1), 940-942(CDRL1-CDRL3 of Ab 5.76.1), 948-950 (CDRL1-CDRL3 of Ab5.77.1), 956-958(CDRL1-CDRL3 of Ab 5.78.1), 964-966 (CDRL1-CDRL3 of Ab 5.80.1), 972-974(CDRL1-CDRL3 of Ab 5.85.1), 980-982 (CDRL1-CDRL3 of Ab 6.37.5), 988-990(CDRL1-CDRL3 of Ab 6.116.6), 996-998 (CDRL1-CDRL3 of Ab 6.139.5),1004-1006 (CDRL1-CDRL3 of Ab 6.147.4), 823-825 (CDRH1-CDRH3 of Ab11H10Hu), 831-833 (CDRH1-CDRH3 of Ab 11H10Rat), 839-841 (CDRH1-CDRH3 ofAb 2.4.1), 847-849 (CDRH1-CDRH3 of Ab 2.20.1), 855-857 (CDRH1-CDRH3 ofAb 2.37.1), 863-865 (CDRH1-CDRH3 of Ab 2.40.1), 871-873 (CDRH1-CDRH3 ofAb 2.41.1), 879-881 (CDRH1-CDRH3 of Ab 2.47.1), 887-889 (CDRH1-CDRH3 ofAb 5.17.1), 895-897 (CDRH1-CDRH3 of Ab 5.23.1), 903-905 (CDRH1-CDRH3 ofAb 5.25.1), 911-913 (CDRH1-CDRH3 of Ab 531.1), 919-921 (CDRH1-CDRH3 ofAb 5.32.1), 927-929 (CDRH1-CDRH3 of Ab 5.40.1), 935-937 (CDRH1-CDRH3 ofAb 5.65.1), 943-945 (CDRH1-CDRH3 of Ab 5.76.1), 951-953 (CDRH1-CDRH3 ofAb 5.77.1), 959-961 (CDRH1-CDRH3 of Ab 5.78.1), 967-969 (CDRH1-CDRH3 ofAb 5.80.1), 975-977 (CDRH1-CDRH3 of Ab5.85.1), 983-985 (CDRH1-CDRH3 ofAb 6.37.5), 991-993 (CDRH1-CDRH3 of Ab 6.116.6), 999-1001 (CDRH1-CDRH3of Ab 6.139.5) and 1007-1009 (CDRH1-CDRH3 of Ab 6.147.4). It iscontemplated that the heterodimeric antibody can include two or moreCDRs from a single antibody, or two or more CDRs from any combination ofthe DKK1 antibodies described herein. Some DKK1 binding componentsinclude both the light chain CDR3 and the heavy chain CDR3. Certain DKK1binding components have variant forms of the CDRs set forth in SEQ IDNOs: 820-822, 828-830, 836-838, 844-846, 852-854, 860-862, 868-869,876-878, 884-886, 892-894, 900-902, 908-910, 916-918, 925-927, 932-934,940-942, 948-950, 956-958, 964-966, 972-974, 980-982, 988-990, 996-998,1004-1006, 823-825, 831-833, 839-841, 847-849, 855-857, 863-865,871-873, 879-881, 887-889, 897-897, 903-905, 911-913, 919-921, 927-929,935-937, 943-945, 951-953, 959-961, 967-969, 975-977, 983-985, 991-993,999-1001 and 1007-1009, with one or more (i.e., 2, 3, 4, 5 or 6) of theCDRs each having at least 80%, 85%, 90% or 95% sequence identity to aCDR sequence set forth in SEQ ID NOs: 820-822, 828-830, 836-838,844-846, 852-854, 860-862, 868-869, 876-878, 884-886, 892-894, 900-902,908-910, 916-918, 925-927, 932-934, 940-942, 948-950, 956-958, 964-966,972-974, 980-982, 988-990, 996-998, 1004-1006, 823-825, 831-833,839-841, 847-849, 855-857, 863-865, 871-873, 879-881, 887-889, 897-897,903-905, 911-913, 919-921, 927-929, 935-937, 943-945, 951-953, 959-961,967-969, 975-977, 983-985, 991-993, 999-1001 and 1007-1009. For example,the DKK1 binding components of the heterodimeric antibody can includeboth a light chain CDR3 and a heavy chain CDR3 that each have at least80%, 85%, 90% or 95% sequence identity to a light chain CDR3 sequenceselected from the group consisting of SEQ ID NOs: 822, 830, 838, 846,854, 862, 870, 878, 886, 894, 902, 910, 918, 926, 934, 942, 950, 958,966, 974, 982, 990, 998 and 1006; and have at least 80%, 85%, 90% or 95%sequence identity to a heavy chain CDR3 sequence selected from the groupconsisting of 825, 833, 841, 849, 857, 865, 873, 881, 889, 897, 905,913, 921, 929, 937, 945, 953, 961, 969, 977, 985, 993, 1001 and 1009.

The CDR sequences of some of the DKK1 binding components that areprovided may also differ from the CDR sequences set forth in SEQ ID NOs:820-822, 828-830, 836-838, 844-846, 852-854, 860-862, 868-869, 876-878,884-886, 892-894, 900-902, 908-910, 916-918, 925-927, 932-934, 940-942,948-950, 956-958, 964-966, 972-974, 980-982, 988-990, 996-998,1004-1006, 823-825, 831-833, 839-841, 847-849, 855-857, 863-865,871-873, 879-881, 887-889, 897-897, 903-905, 911-913, 919-921, 927-929,935-937, 943-945, 951-953, 959-961, 967-969, 975-977, 983-985, 991-993,999-1001 and 1007-1009 such that the amino acid sequence for any givenCDR differs by no more than 1, 2, 3, 4 or 5 amino acid residues.Differences from the listed sequences are typically, but not limited to,conservative substitutions.

In other embodiments, the portion of the heterodimeric molecule thatbinds to DKK1 is selected from those DKK1 binding molecules disclosed inU.S. Pat. No. 7,709,611, U.S. Patent Publ. No. 2008/0193449, U.S. Pat.No. 7,642,238, U.S. Pat. No. 7,700,101, and WO 2007/084344, thedisclosure of all of which are incorporated herein by reference in theirentireties.

Polynucleotides Encoding Engineered Heavy or Light Chains

Encompassed within the invention are nucleic acids encoding heavy and/orlight chain constant and/or variable domains described herein. Nucleicacid molecules of the invention include DNA and RNA in bothsingle-stranded and double-stranded form, as well as the correspondingcomplementary sequences. DNA includes, for example, cDNA, genomic DNA,chemically synthesized DNA, DNA amplified by PCR, and combinationsthereof. The nucleic acid molecules of the invention include full-lengthgenes or cDNA molecules as well as a combination of fragments thereof.The nucleic acids of the invention are preferentially derived from humansources, but the invention includes those derived from non-humanspecies, as well.

An “isolated nucleic acid” is a nucleic acid that has been separatedfrom adjacent genetic sequences present in the genome of the organismfrom which the nucleic acid was isolated, in the case of nucleic acidsisolated from naturally-occurring sources. In the case of nucleic acidssynthesized enzymatically from a template or chemically, such as PCRproducts, cDNA molecules, or oligonucleotides for example, it isunderstood that the nucleic acids resulting from such processes areisolated nucleic acids. An isolated nucleic acid molecule refers to anucleic acid molecule in the form of a separate fragment or as acomponent of a larger nucleic acid construct. In one preferredembodiment, the nucleic acids are substantially free from contaminatingendogenous material. The nucleic acid molecule has preferably beenderived from DNA or RNA isolated at least once in substantially pureform and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical methods (such as those outlined in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences arepreferably provided and/or constructed in the form of an open readingframe uninterrupted by internal non-translated sequences, or introns,that are typically present in eukaryotic genes. Sequences ofnon-translated DNA can be present 5′ or 3′ from an open reading frame,where the same do not interfere with manipulation or expression of thecoding region.

The present invention also includes nucleic acids that hybridize undermoderately stringent conditions, and more preferably highly stringentconditions, to nucleic acids encoding polypeptides as described herein.The basic parameters affecting the choice of hybridization conditionsand guidance for devising suitable conditions are set forth by Sambrook,Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters9 and 11; and Current Protocols in Molecular Biology, 1995, Ausubel etal., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and canbe readily determined by those having ordinary skill in the art basedon, for example, the length and/or base composition of the DNA. One wayof achieving moderately stringent conditions involves the use of aprewashing solution containing 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0),hybridization buffer of about 50% formamide, 6×SSC, and a hybridizationtemperature of about 55 degrees C. (or other similar hybridizationsolutions, such as one containing about 50% formamide, with ahybridization temperature of about 42 degrees C.), and washingconditions of about 60 degrees C., in 0.5×SSC, 0.1% SDS. Generally,highly stringent conditions are defined as hybridization conditions asabove, but with washing at approximately 68 degrees C., 0.2×SSC, 0.1%SDS. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH.sub.2 PO.sub.4, and 1.25 mMEDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15 mMsodium citrate) in the hybridization and wash buffers; washes areperformed for 15 minutes after hybridization is complete. It should beunderstood that the wash temperature and wash salt concentration can beadjusted as necessary to achieve a desired degree of stringency byapplying the basic principles that govern hybridization reactions andduplex stability, as known to those skilled in the art and describedfurther below (see, e.g., Sambrook et al., 1989). When hybridizing anucleic acid to a target nucleic acid of unknown sequence, the hybridlength is assumed to be that of the hybridizing nucleic acid. Whennucleic acids of known sequence are hybridized, the hybrid length can bedetermined by aligning the sequences of the nucleic acids andidentifying the region or regions of optimal sequence complementarity.The hybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5 to 10.degrees C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(degrees C.)=2(# of A+T bases)+4(# of #G+C bases). For hybrids above 18base pairs in length, Tm (degrees C.)=81.5+16.6(log 10 [Na+])+0.41(%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] isthe concentration of sodium ions in the hybridization buffer ([Na+] for1×SSC=0.165M). Preferably, each such hybridizing nucleic acid has alength that is at least 15 nucleotides (or more preferably at least 18nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, orat least 30 nucleotides, or at least 40 nucleotides, or most preferablyat least 50 nucleotides), or at least 25% (more preferably at least 50%,or at least 60%, or at least 70%, and most preferably at least 80%) ofthe length of the nucleic acid of the present invention to which ithybridizes, and has at least 60% sequence identity (more preferably atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, and most preferably at least 99.5%) with thenucleic acid of the present invention to which it hybridizes, wheresequence identity is determined by comparing the sequences of thehybridizing nucleic acids when aligned so as to maximize overlap andidentity while minimizing sequence gaps as described in more detailabove.

Variants are ordinarily prepared by site specific mutagenesis ofnucleotides in the DNA encoding the polypeptide, using cassette or PCRmutagenesis or other techniques well known in the art, to produce DNAencoding the variant, and thereafter expressing the recombinant DNA incell culture as outlined herein. However, antibodies or antibodyfragments comprising variant CDRs having up to about 100-150 residuesmay be prepared by in vitro synthesis using established techniques. Thevariants typically exhibit the same qualitative biological activity asthe naturally occurring analogue, e.g., binding to antigen, althoughvariants can also be selected which have modified characteristics aswill be more fully outlined herein.

As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids may be made,all of which encode the CDRs (and heavy and light chains or othercomponents of a heterodimeric antibody described herein) of theinvention. Thus, having identified a particular amino acid sequence,those skilled in the art could make any number of different nucleicacids, by simply modifying the sequence of one or more codons in a waywhich does not change the amino acid sequence of the encoded protein.

The invention also provides expression systems and constructs in theform of plasmids, expression vectors, transcription or expressioncassettes which comprise at least one polynucleotide as above. Inaddition, the invention provides host cells comprising such expressionsystems or constructs.

Typically, expression vectors used in the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences,” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the polypeptidecoding sequence; the oligonucleotide sequence encodes polyHis (such ashexaHis), or another “tag” such as FLAG, HA (hemaglutinin influenzavirus), or myc, for which commercially available antibodies exist. Thistag is typically fused to the polypeptide upon expression of thepolypeptide, and can serve as a means for affinity purification ordetection of the polypeptide from the host cell. Affinity purificationcan be accomplished, for example, by column chromatography usingantibodies against the tag as an affinity matrix. Optionally, the tagcan subsequently be removed from the purified polypeptide by variousmeans such as using certain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence may be known. Here, the flanking sequence may be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

Whether all or only a portion of the flanking sequence is known, it maybe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence may be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationmay be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Specific selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. Advantageously, a neomycin resistance genemay also be used for selection in both prokaryotic and eukaryotic hostcells.

Other selectable genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thyrnidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as anantibody light or heavy chain. As a result, increased quantities of apolypeptide are synthesized from the amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed. In certain embodiments, one or more coding regions may beoperably linked to an internal ribosome binding site (IRES), allowingtranslation of two open reading frames from a single RNA transcript.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orprosequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addprosequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning vectors of the invention will typically contain apromoter that is recognized by the host organism and operably linked tothe molecule encoding the polypeptide. Promoters are untranscribedsequences located upstream (i.e., 5′) to the start codon of a structuralgene (generally within about 100 to 1000 bp) that control transcriptionof the structural gene. Promoters are conventionally grouped into one oftwo classes: inducible promoters and constitutive promoters. Induciblepromoters initiate increased levels of transcription from DNA undertheir control in response to some change in culture conditions, such asthe presence or absence of a nutrient or a change in temperature.Constitutive promoters, on the other hand, uniformly transcribe gene towhich they are operably linked, that is, with little or no control overgene expression. A large number of promoters, recognized by a variety ofpotential host cells, are well known. A suitable promoter is operablylinked to the DNA encoding e.g., heavy chain or light chain, by removingthe promoter from the source DNA by restriction enzyme digestion andinserting the desired promoter sequence into the vector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest include, but are notlimited to: SV40 early promoter (Benoist and Chambon, 1981, Nature290:304-310); CMV promoter (Thomsen et al., 1984, Proc. Natl. Acad.U.S.A. 81:659-663); the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797);herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences from themetallothionine gene Prinster et al., 1982, Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulingene control region that is active in pancreatic beta cells (Hanahan,1985, Nature 315:115-122); the immunoglobulin gene control region thatis active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol.Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-495); the albumin gene control region that isactive in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science253:53-58); the alpha 1-antitrypsin gene control region that is activein liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, 1985, Nature 314:283-286); and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

An enhancer sequence may be inserted into the vector to increasetranscription of DNA encoding light chain or heavy chain of theinvention by higher eukaryotes. Enhancers are cis-acting elements ofDNA, usually about 10-300 by in length, that act on the promoter toincrease transcription. Enhancers are relatively orientation andposition independent, having been found at positions both 5′ and 3′ tothe transcription unit. Several enhancer sequences available frommammalian genes are known (e.g., globin, elastase, albumin,alpha-feto-protein and insulin). Typically, however, an enhancer from avirus is used. The SV40 enhancer, the cytomegalovirus early promoterenhancer, the polyoma enhancer, and adenovirus enhancers known in theart are exemplary enhancing elements for the activation of eukaryoticpromoters. While an enhancer may be positioned in the vector either 5′or 3′ to a coding sequence, it is typically located at a site 5′ fromthe promoter. A sequence encoding an appropriate native or heterologoussignal sequence (leader sequence or signal peptide) can be incorporatedinto an expression vector, to promote extracellular secretion of theantibody. The choice of signal peptide or leader depends on the type ofhost cells in which the antibody is to be produced, and a heterologoussignal sequence can replace the native signal sequence. Examples ofsignal peptides that are functional in mammalian host cells include thefollowing: the signal sequence for interleukin-7 (IL-7) described inU.S. Pat. No. 4,965,195; the signal sequence for interleukin-2 receptordescribed in Cosman et al., 1984, Nature 312:768; the interleukin-4receptor signal peptide described in EP Patent No. 0367 566; the type Iinterleukin-1 receptor signal peptide described in U.S. Pat. No.4,968,607; the type II interleukin-1 receptor signal peptide describedin EP Patent No. 0 460 846.

The vector may contain one or more elements that facilitate expressionwhen the vector is integrated into the host cell genome. Examplesinclude an EASE element (Aldrich et al. 2003 Biotechnol Prog.19:1433-38) and a matrix attachment region (MAR). MARs mediatestructural organization of the chromatin and may insulate the integratedvactor from “position” effect. Thus, MARs are particularly useful whenthe vector is used to create stable transfectants. A number of naturaland synthetic MAR-containing nucleic acids are known in the art, e.g.,U.S. Pat. Nos. 6,239,328; 7,326,567; 6,177,612; 6,388,066; 6,245,974;7,259,010; 6,037,525; 7,422,874; 7,129,062.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe flanking sequences described herein are not already present in thevector, they may be individually obtained and ligated into the vector.Methods used for obtaining each of the flanking sequences are well knownto one skilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding light chain, a heavy chain, or a light chain and a heavy chainsequence has been inserted into the proper site of the vector, thecompleted vector may be inserted into a suitable host cell foramplification and/or polypeptide expression. The transformation of anexpression vector into a selected host cell may be accomplished by wellknown methods including transfection, infection, calcium phosphateco-precipitation, electroporation, microinjection, lipofection,DEAE-dextran mediated transfection, or other known techniques. Themethod selected will in part be a function of the type of host cell tobe used. These methods and other suitable methods are well known to theskilled artisan, and are set forth, for example, in Sambrook et al.,2001, supra.

A host cell, when cultured under appropriate conditions, synthesizesheterodimeric antibody that can subsequently be collected from theculture medium (if the host cell secretes it into the medium) ordirectly from the host cell producing it (if it is not secreted). Theselection of an appropriate host cell will depend upon various factors,such as desired expression levels, polypeptide modifications that aredesirable or necessary for activity (such as glycosylation orphosphorylation) and ease of folding into a biologically activemolecule. A host cell may be eukaryotic or prokaryotic.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC) and any celllines used in an expression system known in the art can be used to makethe recombinant polypeptides of the invention. In general, host cellsare transformed with a recombinant expression vector that comprises DNAencoding a desired heterodimeric antibody. Among the host cells that maybe employed are prokaryotes, yeast or higher eukaryotic cells.Prokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. Higher eukaryotic cells include insect cellsand established cell lines of mammalian origin. Examples of suitablemammalian host cell lines include the COS-7 line of monkey kidney cells(ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells,C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells,or their derivatives such as Veggie CHO and related cell lines whichgrow in serum-free media (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cellline derived from the African green monkey kidney cell line CV1 (ATCCCCL 70) as described by McMahan et al., 1991, EMBO J. 10: 2821, humanembryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermalA431 cells, human Colo205 cells, other transformed primate cell lines,normal diploid cells, cell strains derived from in vitro culture ofprimary tissue, primary explants, HL-60, U937, HaK or Jurkat cells.Optionally, mammalian cell lines such as HepG2/3B, KB, NIH 3T3 or S49,for example, can be used for expression of the polypeptide when it isdesirable to use the polypeptide in various signal transduction orreporter assays. Alternatively, it is possible to produce thepolypeptide in lower eukaryotes such as yeast or in prokaryotes such asbacteria. Suitable yeasts include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous polypeptides. Suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous polypeptides.

If the antibody or fragment is made in yeast or bacteria, it may bedesirable to modify the product produced therein, for example byphosphorylation or glycosylation of the appropriate sites, in order toobtain a functional product. Such covalent attachments can beaccomplished using known chemical or enzymatic methods. A polypeptidecan also be produced by operably linking the isolated nucleic acid ofthe invention to suitable control sequences in one or more insectexpression vectors, and employing an insect expression system. Materialsand methods for baculovirus/insect cell expression systems arecommercially available in kit form from, e.g., Invitrogen, San Diego,Calif., U.S.A. (the MaxBac□ kit), and such methods are well known in theart, as described in Summers and Smith, Texas Agricultural ExperimentStation Bulletin No. 1555 (1987), and Luckow and Summers, Bio/Technology6:47 (1988). Cell-free translation systems could also be employed toproduce polypeptides, such as antibodies or fragments, using RNAsderived from nucleic acid constructs disclosed herein. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described by Pouwels et al. (CloningVectors: A Laboratory Manual, Elsevier, New York, 1985). A host cellthat comprises an isolated nucleic acid of the invention, preferablyoperably linked to at least one expression control sequence, is a“recombinant host cell”.

In certain embodiments, cell lines may be selected through determiningwhich cell lines have high expression levels and constitutively produceantigen binding proteins with the desired binding properties. In anotherembodiment, a cell line from the B cell lineage that does not make itsown antibody but has a capacity to make and secrete a heterologousantibody can be selected.

Therapeutic Methods

The heterodimeric antibody molecules described herein are useful fortreating or preventing bone-related disorders, such as bone-relateddisorders associated with abnormal osteoblast or osteoclast activity. Insome embodiments, the heterodimeric antibody is administered to asubject suffering from a bone related disorder selected from the groupconsisting of achondroplasia, cleidocranial dysostosis,enchondromatosis, fibrous dysplasia, Gaucher's Disease, hypophosphatemicrickets, Marfan's syndrome, multiple hereditary exotoses,neurofibromatosis, osteogenesis imperfecta, osteopetrosis,osteopoikilosis, sclerotic lesions, pseudoarthrosis, pyogenicosteomyelitis, periodontal disease, anti-epileptic drug induced boneloss, primary and secondary hyperparathyroidism, familialhyperparathyroidism syndromes, weightlessness induced bone loss,osteoporosis in men, postmenopausal bone loss, osteoarthritis, renalosteodystrophy, infiltrative disorders of bone, oral bone loss,osteonecrosis of the jaw, juvenile Paget's disease, melorheostosis,metabolic bone diseases, mastocytosis, sickle cell anemia/disease, organtransplant related bone loss, kidney transplant related bone loss,systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenilearthritides, thalassemia, mucopolysaccharidoses, Fabry Disease, TurnerSyndrome, Down Syndrome, Klinefelter Syndrome, leprosy, Perthe'sDisease, adolescent idiopathic scoliosis, infantile onset multi-systeminflammatory disease, Winchester Syndrome, Menkes Disease, Wilson'sDisease, ischemic bone disease (such as Legg-Calve-Perthes disease andregional migratory osteoporosis), anemic states, conditions caused bysteroids, glucocorticoid-induced bone loss, heparin-induced bone loss,bone marrow disorders, scurvy, malnutrition, calcium deficiency,osteoporosis, osteopenia, alcoholism, chronic liver disease,postmenopausal state, chronic inflammatory conditions, rheumatoidarthritis, inflammatory bowel disease, ulcerative colitis, inflammatorycolitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy-relatedbone loss, diabetes mellitus, hyperthyroidism, thyroid disorders,parathyroid disorders, Cushing's disease, acromegaly, hypogonadism,immobilization or disuse, reflex sympathetic dystrophy syndrome,regional osteoporosis, osteomalacia, bone loss associated with jointreplacement, HIV associated bone loss, bone loss associated with loss ofgrowth hormone, bone loss associated with cystic fibrosis,chemotherapy-associated bone loss, tumor-induced bone loss,cancer-related bone loss, hormone ablative bone loss, multiple myeloma,drug-induced bone loss, anorexia nervosa, disease-associated facial boneloss, disease-associated cranial bone loss, disease-associated bone lossof the jaw, disease-associated bone loss of the skull, bone lossassociated with aging, facial bone loss associated with aging, cranialbone loss associated with aging, jaw bone loss associated with aging,skull bone loss associated with aging, and bone loss associated withspace travel.

In some embodiments, the heterodimeric antibodies described herein areuseful for improving outcomes in orthopedic procedures, dentalprocedures, implant surgery, joint replacement, bone grafting, bonecosmetic surgery and bone repair such as fracture healing, nonunionhealing, delayed union healing and facial reconstruction. A compositioncomprising one or more heterodimeric antibodies or fragments may beadministered before, during and/or after the procedure, replacement,graft, surgery or repair.

The heterodimeric antibody need not cure the subject of the disorder orcompletely protect against the onset of a bone-related disorder toachieve a beneficial biological response. The heterodimeric antibody maybe used prophylactically, meaning to protect, in whole or in part,against a bone-related disorder or symptom thereof. The heterodimericantibody also may be used therapeutically to ameliorate, in whole or inpart, a bone-related disorder or symptom thereof, or to protect, inwhole or in part, against further progression of a bone-related disorderor symptom thereof. Indeed, the materials and methods of the inventionare particularly useful for increasing bone mineral density andmaintaining the increased bone mineral density over a period of time.

In some embodiments, one or more administrations of a heterodimericantibody described herein are carried out over a therapeutic period of,for example, about 1 week to about 18 months (e.g., about 1 month toabout 12 months, about 1 month to about 9 months or about 1 month toabout 6 months or about 1 month to about 3 months). In some embodiments,a subject is administered one or more doses of a heterodimeric antibodydescribed herein over a therapeutic period of, for example about 1 monthto about 12 months (52 weeks) (e.g., about 2 months, about 3 months,about 4 months, about 5 months, about 6 months, about 7 months, about 8months, about 9 months, about 10 months, or about 11 months). In someembodiments, a subject is administered one or more doses of theheterodimeric antibody to maintain bone mineral density. The term“maintain bone mineral density” as used herein means that the increasedbone mineral density resulting from the initial dose of theheterodimeric antibody does not fall more than about 1% to about 5% overthe course of about 6 months, about 9 months about 1 year, about 18months, about 2 years, or over the course of the patient's life). Itwill be appreciated that a patient can require alternate treatmentphases for increasing bone density and maintaining bone density.

In addition, it may be advantageous to administer multiple doses of theheterodimeric antibody or space out the administration of doses,depending on the therapeutic regimen selected for a particular subject.In some embodiments, the heterodimeric antibody or fragment thereof isadministered periodically over a time period of one year (12 months, 52weeks) or less (e.g., 9 months or less, 6 months or less, or 3 months orless). In this regard, the heterodimeric antibody or fragment thereof isadministered to the human once every about 3 days, or about 7 days, or 2weeks, or 3 weeks, or 4 weeks, or 5 weeks, or 6 weeks, or 7 weeks, or 8weeks, or 9 weeks, or 10 weeks, or 11 weeks, or 12 weeks, or 13 weeks,or 14 weeks, or 15 weeks, or 16 weeks, or 17 weeks, or 18 weeks, or 19weeks, or 20 weeks, or 21 weeks, or 22 weeks, or 23 weeks, or 6 months,or 12 months.

In some embodiments, one or more doses of the heterodimeric antibody orfragment thereof are administered in an amount and for a time effectiveto treat a bone disorder associated with decreased bone mineral density.In various embodiments, one or more doses comprising from about 50milligrams to about 1,000 milligrams of the heterodimeric antibody areadministered per week to a subject (e.g., a human subject). For example,a dose of heterodimeric antibody can comprise at least about 5 mg, 15mg, 25 mg, 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg,about 100 mg, about 120 mg, about 150 mg, about 200 mg, about 240 mg,about 250 mg, about 280 mg, about 300 mg, about 350 mg, about 400 mg,about 420 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg,about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg,about 900 mg, about 950 mg or up to about 1,000 mg of heterodimericantibody. Ranges between any and all of these endpoints are alsocontemplated, e.g. about 50 mg to about 80 mg, about 70 mg to about 140mg, about 70 mg to about 270 mg, about 75 mg to about 100 mg, about 100mg to about 150 mg, about 140 mg to about 210 mg, or about 150 mg toabout 200 mg, or about 180 mg to about 270 mg, or about 280 to about 410mg. The dose is administered at any interval, such as multiple times aweek (e.g., twice or three times per week), once a week, once every twoweeks, once every three weeks, or once every four weeks. In some or anyembodiments, a dose of heterodimeric antibody ranging from about 120 mgto about 210 mg is administered twice a week. In some or anyembodiments, a dose of about 140 mg of the heterodimeric antibody isadministered twice a week.

In some embodiments, the one or more doses of heterodimeric antibody cancomprise between about 0.1 to about 50 milligrams (e.g., between about 5and about 50 milligrams), or about 1 to about 100 milligrams, ofheterodimeric antibody per kilogram of body weight (mg/kg). For example,the dose of heterodimeric antibody may comprise at least about 0.1mg/kg, 0.5 mg/kg, 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg,about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9mg/kg, about 10 mg/kg, about 20 mg/kg, about 25 mg/kg, about 26 mg/kg,about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, about 30 mg/kg, about 31mg/kg, about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35 mg/kg,about 36 mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg, about 40mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg, about 44 mg/kg,about 45 mg/kg, about 46 mg/kg, about 47 mg/kg, about 48 mg/kg, or about49 mg/kg, or about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg,about 90 mg/kg, about 95 mg/kg, or up to about 100 mg/kg. Ranges betweenany and all of these endpoints are also contemplated, e.g., about 1mg/kg to about 3 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg toabout 8 mg/kb, about 3 mg/kg to about 8 mg·kg, about 1 mg/kg to about 10mg/kg, about 1 mg/kg to about 20 mg/kg, about 1 mg/kg to about 40 mg/kg,about 5 mg/kg to about 30 mg/kg, or about 5 mg/kg to about 20 mg/kg.

Monitoring Therapy

Heterodimeric antibody-mediated increases in bone mineral content orbone density may be measured using single- and dual-energy X-rayabsorptometry, ultrasound, computed tomography, radiography, andmagnetic resonance imaging. The amount of bone mass may also becalculated from body weights or by using other methods (seeGuinness-Hey, Metab. Bone Dis. Relat. Res., 5:177-181 (1984)). Animalmodels are used in the art for testing the effect of the pharmaceuticalcompositions and methods on, for example, parameters of bone loss, boneresorption, bone formation, bone strength, or bone mineralization thatmimic conditions of human disease such as osteoporosis and osteopenia.Examples of such models include the ovariectomized rat model (Kalu, Boneand Mineral, 15:175-192 (1991); Frost and Jee, Bone and Mineral,18:227-236 (1992); and Jee and Yao, J. Musculoskel. Neuron. Interact.,1:193-207 (2001)). The methods for measuring heterodimeric antibodyactivity described herein also may be used to determine the efficacy ofother sclerostin inhibitors.

In humans, bone mineral density can be determined clinically using dualx-ray absorptiometry (DXA) of, for example, the hip and spine. Othertechniques include quantitative computed tomography (QCT),ultrasonography, single-energy x-ray absorptiometry (SXA), andradiographic absorptiometry. Common central skeletal sites formeasurement include the spine and hip; peripheral sites include theforearm, finger, wrist and heel. Except for ultrasonography, theAmerican Medical Association notes that BMD techniques typically involvethe use of x-rays and are based on the principle that attenuation of theradiation depends on thickness and composition of the tissues in theradiation path. All techniques involve the comparison of results to anormative database.

Alternatively, a physiological response to one or more sclerostinbinding agents can be gauged by monitoring bone marker levels. Bonemarkers are products created during the bone remodeling process and arereleased by bone, osteoblasts, and/or osteoclasts. Fluctuations in boneresorption and/or bone formation “marker” levels imply changes in boneremodeling/modeling. The International Osteoporosis Foundation (IOF)recommends using bone markers to monitor bone density therapies (see,e.g., Delmas et al., Osteoporos Int., Suppl. 6:S2-17 (2000),incorporated herein by reference). Markers indicative of bone resorption(or osteoclast activity) include, for example, C-telopeptide (e.g.,C-terminal telopeptide of type 1 collagen (CTX) or serum cross-linkedC-telopeptide), N-telopeptide (N-terminal telopeptide of type 1 collagen(NTX)), deoxypyridinoline (DPD), pyridinoline, urinary hydroxyproline,galactosyl hydroxylysine, and tartrate-resistant acid phosphatase (e.g.,serum tartrate-resistant acid phosphatase isoform 5b). Boneformation/mineralization markers include, but are not limited to,bone-specific alkaline phosphatase (BSAP), peptides released from N- andC-terminal extension of type I procollagen (P1NP, PICP), and osteocalcin(OstCa). Several kits are commercially-available to detect and quantifymarkers in clinical samples, such as urine and blood.

Combination Therapy

Treatment of a pathology by combining two or more agents that target thesame pathogen or biochemical pathway or biological process sometimesresults in greater efficacy and diminished side effects relative to theuse of a therapeutically relevant dose of each agent alone. In somecases, the efficacy of the drug combination is additive (the efficacy ofthe combination is approximately equal to the sum of the effects of eachdrug alone), but in other cases the effect is synergistic (the efficacyof the combination is greater than the sum of the effects of each druggiven alone). As used herein, the term “combination therapy” means thattwo or more agents are delivered in a simultaneous manner, e.g.,concurrently, or wherein one of the agents is administered first,followed by the second agent, e.g., sequentially.

In some embodiments, the heterodimeric antibody is administered alongwith a standard of care therapeutic for the treatment of decreased bonemineral density (i.e., the heterodimeric antibody and standard of caretherapeutic are part of the same treatment plan). As used herein, theterm “standard of care” refers to a treatment that is generally acceptedby clinicians for a certain type of patient diagnosed with a type ofillness. In some embodiments, the heterodimeric antibody is administeredalong with a second bone-enhancing agent useful for the treatment ofdecreased bone mineral density or bone defect. In some embodiments, thebone-enhancing agent is selected from the group consisting of ananti-resorptive agent, a bone-forming agent (i.e., anabolic), anestrogen receptor modulator (including, but not limited to, raloxifene,bazedoxifene and lasofoxifene) and a drug that has an inhibitory effecton osteoclasts. In some embodiments, the second bone-enhancing agent isselected from the group consisting of a bisphosphonate (including, butnot limited to, alendronate sodium (FOSAMAX®), risedronate, ibandronatesodium (BONIVA®) and zoledronic acid (RECLAST®)); an estrogen orestrogen analogue; an anti-RANK ligand (RANKL) inhibitor, such as ananti-RANKL antibody (e.g., PROLIA®); vitamin D, or a vitamin Dderivative or mimic thereof; a calcium source, a cathepsin-K (cat-K)inhibitor (e.g. odanacatib), Tibolone, calcitonin or a calcitriol; andhormone replacement therapy. In some embodiments, the secondbone-enhancing agent includes, but is not limited to, parathyroidhormone (PTH) or a peptide fragment thereof, PTH-related protein(PTHrp), bone morphogenetic protein, osteogenin, NaF, a PGE2 agonist, astatin, strontium ranelate, a sclerostin inhibitor (e.g., ananti-sclerostin antibody described in, for example, U.S. Pat. No.7,592,429 or 7,872,106), and an anti-DKK1 antibody or inhibitor. In someembodiments, the second bone-enhancing agent is Forteo® (Teriparatide),Preotact®, or Protelos®.

In some embodiments, the combination therapy employing a heterodimericantibody described herein may precede or follow administration ofadditional therapeutic(s) (e.g., second bone-enhancing agent) byintervals ranging from minutes to weeks to months. For example, separatemodalities are administered within about 24 hours of each other, e.g.,within about 6-12 hours of each other, or within about 1-2 hours of eachother, or within about 10-30 minutes of each other. In some situations,it may be desirable to extend the time period for treatmentsignificantly, where several days (2, 3, 4, 5, 6 or 7 days) to severalweeks (1, 2, 3, 4, 5, 6, 7 or 8 weeks) lapse between the respectiveadministrations of different modalities. Repeated treatments with one orboth agents/therapies of the combination therapy is specificallycontemplated.

Maintenance Therapeutic Regimen

Also contemplated is the use of a second bone-enhancing agent and/orheterodimeric antibody described herein in a maintenance regimen to,e.g., prevent or slow the loss of bone mineral density. In this regard,a method or use described herein optionally comprises administering oneor more amounts of a second bone-enhancing agent effective to maintainbone mineral density for a maintenance period of about 1 week to about 5years after the treatment period with the heterodimeric antibody hasended. For example, in some embodiments, a method or use describedherein comprises the administration of a second bone-enhancing agent tothe subject for a maintenance period of about at least about 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10weeks, 11 weeks, 12 weeks, 3 months, 13 weeks, 14 weeks, 15 weeks, 16weeks, 4 months, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 5 months, 21weeks, 22 weeks, 23 weeks, 24 weeks, 6 months, 25 weeks, 26 weeks, 27weeks 28 weeks, 7 months, 29 weeks, 30 weeks, 31 weeks or longer (e.g.,8 months, 9 months, 10 months, 11 months, 1 year, 15 months, 18 months,2 years, 3 years, 4 years, 5 years or longer (e.g., over the lifetime ofthe subject). In some embodiments, the maintenance period is about 6-12weeks. In some embodiments, the maintenance period is about 4-12 weeks,or about 1-3 months. In some embodiments, the maintenance period isabout 12-20 weeks, or about 3-5 months. In some embodiments, themaintenance period is about 20-32 weeks, or about 5-8 months. In someembodiments, the maintenance period is about 24-36 weeks, or about 6-9months. In some embodiments, the maintenance period is about 1 year,about 2 years, about 3 years, about 4 years, about 5 years or longer.“Maintaining” bone mineral density includes maintaining similar levelsof bone mineral density parameters experienced in the subject thatreceived the heterodimeric antibody treatment.

Similarly, a method or use described herein optionally comprisessubsequently administering one or more amounts of a heterodimericantibody effective to maintain bone mineral density for a maintenanceperiod of at least about least about 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12weeks, 3 months, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 4 months, 17weeks, 18 weeks, 19 weeks, 20 weeks, 5 months, 21 weeks, 22 weeks, 23weeks, 24 weeks, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years orlonger (e.g., over the lifetime of the subject) after the treatmentperiod has ended. In some embodiments, the maintenance period is about6-12 weeks. In some embodiments, the maintenance period is about 4-12weeks, or about 1-3 months. In some embodiments, the maintenance periodis about 12-20 weeks, or about 3-5 months. In some embodiments, themaintenance period is about 20-32 weeks, or about 5-8 months. In someembodiments, the maintenance period is about 24-36 weeks, or about 6-9months. In some embodiments, the maintenance period is about 1 year,about 2 year, about 3 years, about 4 years, about 5 years or longer.

Pharmaceutical Compositions

In some embodiments, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of one or a plurality ofthe antigen binding proteins of the invention together with apharmaceutically effective diluents, carrier, solubilizer, emulsifier,preservative, and/or adjuvant. Pharmaceutical compositions of theinvention include, but are not limited to, liquid, frozen, andlyophilized compositions.

Preferably, formulation materials are nontoxic to recipients at thedosages and concentrations employed. In specific embodiments,pharmaceutical compositions comprising a therapeutically effectiveamount of heterodimeric antibody or fragment are provided.

In some embodiments, the pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine, proline, or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates or other organic acids); bulking agents(such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. See,REMINGTON'S PHARMACEUTICAL SCIENCES, 18″ Edition, (A. R. Genrmo, ed.),1990, Mack Publishing Company.

In some embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theheterodimeric antibody or fragment. In certain embodiments, the primaryvehicle or carrier in a pharmaceutical composition may be either aqueousor non-aqueous in nature. For example, a suitable vehicle or carrier maybe water for injection, physiological saline solution or artificialcerebrospinal fluid, possibly supplemented with other materials commonin compositions for parenteral administration. Neutral buffered salineor saline mixed with serum albumin are further exemplary vehicles. Inspecific embodiments, pharmaceutical compositions comprise Tris bufferof about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and mayfurther include sorbitol or a suitable substitute therefor. In certainembodiments of the invention, the composition may be prepared forstorage by mixing the selected composition having the desired degree ofpurity with optional formulation agents (REMINGTON'S PHARMACEUTICALSCIENCES, supra) in the form of a lyophilized cake or an aqueoussolution. Further, in some embodiments, the heterodimeric antibody orfragment may be formulated as a lyophilizate using appropriateexcipients such as sucrose.

The pharmaceutical compositions of the invention can be selected forparenteral delivery. Alternatively, the compositions may be selected forinhalation or for delivery through the digestive tract, such as orally.Preparation of such pharmaceutically acceptable compositions is withinthe skill of the art. The formulation components are present preferablyin concentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be provided in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired heterodimeric antibody or fragment in a pharmaceuticallyacceptable vehicle. A particularly suitable vehicle for parenteralinjection is sterile distilled water in which the heterodimeric antibodyor fragment is formulated as a sterile, isotonic solution, properlypreserved. In certain embodiments, the preparation involves theformulation of the desired molecule with an agent, such as injectablemicrospheres, bio-erodible particles, polymeric compounds (such aspolylactic acid or polyglycolic acid), beads or liposomes, that mayprovide controlled or sustained release of the product which can bedelivered via depot injection. In certain embodiments, hyaluronic acidmay also be used, having the effect of promoting sustained duration inthe circulation. In certain embodiments, implantable drug deliverydevices may be used to introduce the desired heterodimeric antibody orfragment.

Pharmaceutical compositions of the invention can be formulated forinhalation. In these embodiments, heterodimeric antibody or fragment isadvantageously formulated as a dry, inhalable powder. In specificembodiments, heterodimeric antibody or fragment inhalation solutions mayalso be formulated with a propellant for aerosol delivery. In certainembodiments, solutions may be nebulized. Pulmonary administration andformulation methods therefore are further described in InternationalPatent Application No. PCT/US94/001875, which is incorporated byreference and describes pulmonary delivery of chemically modifiedproteins.

It is also contemplated that formulations can be administered orally.Heterodimeric antibody or fragments that are administered in thisfashion can be formulated with or without carriers customarily used inthe compounding of solid dosage forms such as tablets and capsules. Incertain embodiments, a capsule may be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized. Additional agents can be included to facilitate absorption ofthe heterodimeric antibody or fragment. Diluents, flavorings, lowmelting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders may also be employed.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving antigen binding proteins insustained- or controlled-delivery formulations. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. See,for example, International Patent Application No. PCT/US93/00829, whichis incorporated by reference and describes controlled release of porouspolymeric microparticles for delivery of pharmaceutical compositions.Sustained-release preparations may include semipermeable polymermatrices in the form of shaped articles, e.g., films, or microcapsules.Sustained release matrices may include polyesters, hydrogels,polylactides (as disclosed in U.S. Pat. No. 3,773,919 and EuropeanPatent Application Publication No. EP058481, each of which isincorporated by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater.Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinylacetate (Langer et al., 1981, supra) or poly-D(−)-3-hydroxybutyric acid(European Patent Application Publication No. EP133988). Sustainedrelease compositions may also include liposomes that can be prepared byany of several methods known in the art. See, e.g., Eppstein et al.,1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-3692; European PatentApplication Publication Nos. EP036676; EP088046 and EP143949,incorporated by reference.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method may beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Aspects of the invention includes self-buffering heterodimeric antibodyor fragment formulations, which can be used as pharmaceuticalcompositions, as described in international patent application WO2006/138181A2 (PCT/US2006/022599), which is incorporated by reference inits entirety herein.

As discussed above, certain embodiments provide heterodimeric antibodyor fragment compositions, particularly pharmaceutical heterodimericantibody or fragment compositions, that comprise, in addition to theheterodimeric antibody or fragment, one or more excipients such as thoseillustratively described in this section and elsewhere herein.Excipients can be used in the invention in this regard for a widevariety of purposes, such as adjusting physical, chemical, or biologicalproperties of formulations, such as adjustment of viscosity, and orprocesses of the invention to improve effectiveness and or to stabilizesuch formulations and processes against degradation and spoilage due to,for instance, stresses that occur during manufacturing, shipping,storage, pre-use preparation, administration, and thereafter.

A variety of expositions are available on protein stabilization andformulation materials and methods useful in this regard, such as Arakawaet al., “Solvent interactions in pharmaceutical formulations,” PharmRes. 8(3): 285-91 (1991); Kendrick et al., “Physical stabilization ofproteins in aqueous solution,” in: RATIONAL DESIGN OF STABLE PROTEINFORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds.Pharmaceutical Biotechnology. 13: 61-84 (2002), and Randolph et al.,“Surfactant-protein interactions,” Pharm Biotechnol. 13: 159-75 (2002),each of which is herein incorporated by reference in its entirety,particularly in parts pertinent to excipients and processes of the samefor self-buffering protein formulations in accordance with the currentinvention, especially as to protein pharmaceutical products andprocesses for veterinary and/or human medical uses.

Salts may be used in accordance with certain embodiments of theinvention to, for example, adjust the ionic strength and/or theisotonicity of a formulation and/or to improve the solubility and/orphysical stability of a protein or other ingredient of a composition inaccordance with the invention.

As is well known, ions can stabilize the native state of proteins bybinding to charged residues on the protein's surface and by shieldingcharged and polar groups in the protein and reducing the strength oftheir electrostatic interactions, attractive, and repulsiveinteractions. Ions also can stabilize the denatured state of a proteinby binding to, in particular, the denatured peptide linkages (—CONH) ofthe protein. Furthermore, ionic interaction with charged and polargroups in a protein also can reduce intermolecular electrostaticinteractions and, thereby, prevent or reduce protein aggregation andinsolubility.

Ionic species differ significantly in their effects on proteins. Anumber of categorical rankings of ions and their effects on proteinshave been developed that can be used in formulating pharmaceuticalcompositions in accordance with the invention. One example is theHofmeister series, which ranks ionic and polar non-ionic solutes bytheir effect on the conformational stability of proteins in solution.Stabilizing solutes are referred to as “kosmotropic.” Destabilizingsolutes are referred to as “chaotropic.” Kosmotropes commonly are usedat high concentrations (e.g., >1 molar ammonium sulfate) to precipitateproteins from solution (“salting-out”). Chaotropes commonly are used todenture and/or to solubilize proteins (“salting-in”). The relativeeffectiveness of ions to “salt-in” and “salt-out” defines their positionin the Hofmeister series.

Free amino acids can be used in heterodimeric antibody or fragmentformulations in accordance with various embodiments of the invention asbulking agents, stabilizers, and antioxidants, as well as other standarduses. Lysine, proline, serine, and alanine can be used for stabilizingproteins in a formulation. Glycine is useful in lyophilization to ensurecorrect cake structure and properties. Arginine may be useful to inhibitprotein aggregation, in both liquid and lyophilized formulations.Methionine is useful as an antioxidant.

Polyols include sugars, e.g., mannitol, sucrose, and sorbitol andpolyhydric alcohols such as, for instance, glycerol and propyleneglycol, and, for purposes of discussion herein, polyethylene glycol(PEG) and related substances. Polyols are kosmotropic. They are usefulstabilizing agents in both liquid and lyophilized formulations toprotect proteins from physical and chemical degradation processes.Polyols also are useful for adjusting the tonicity of formulations.

Among polyols useful in select embodiments of the invention is mannitol,commonly used to ensure structural stability of the cake in lyophilizedformulations. It ensures structural stability to the cake. It isgenerally used with a lyoprotectant, e.g., sucrose. Sorbitol and sucroseare among preferred agents for adjusting tonicity and as stabilizers toprotect against freeze-thaw stresses during transport or the preparationof bulks during the manufacturing process. Reducing sugars (whichcontain free aldehyde or ketone groups), such as glucose and lactose,can glycate surface lysine and arginine residues. Therefore, theygenerally are not among preferred polyols for use in accordance with theinvention. In addition, sugars that form such reactive species, such assucrose, which is hydrolyzed to fructose and glucose under acidicconditions, and consequently engenders glycation, also is not amongpreferred polyols of the invention in this regard. PEG is useful tostabilize proteins and as a cryoprotectant and can be used in theinvention in this regard.

Embodiments of the heterodimeric antibody or fragment formulationsfurther comprise surfactants. Protein molecules may be susceptible toadsorption on surfaces and to denaturation and consequent aggregation atair-liquid, solid-liquid, and liquid-liquid interfaces. These effectsgenerally scale inversely with protein concentration. These deleteriousinteractions generally scale inversely with protein concentration andtypically are exacerbated by physical agitation, such as that generatedduring the shipping and handling of a product.

Surfactants routinely are used to prevent, minimize, or reduce surfaceadsorption. Useful surfactants in the invention in this regard includepolysorbate 20, polysorbate 80, other fatty acid esters of sorbitanpolyethoxylates, and poloxamer 188.

Surfactants also are commonly used to control protein conformationalstability. The use of surfactants in this regard is protein-specificsince, any given surfactant typically will stabilize some proteins anddestabilize others.

Polysorbates are susceptible to oxidative degradation and often, assupplied, contain sufficient quantities of peroxides to cause oxidationof protein residue side-chains, especially methionine. Consequently,polysorbates should be used carefully, and when used, should be employedat their lowest effective concentration. In this regard, polysorbatesexemplify the general rule that excipients should be used in theirlowest effective concentrations.

Embodiments of heterodimeric antibody or fragment formulations furthercomprise one or more antioxidants. To some extent deleterious oxidationof proteins can be prevented in pharmaceutical formulations bymaintaining proper levels of ambient oxygen and temperature and byavoiding exposure to light. Antioxidant excipients can be used as wellto prevent oxidative degradation of proteins. Among useful antioxidantsin this regard are reducing agents, oxygen/free-radical scavengers, andchelating agents. Antioxidants for use in therapeutic proteinformulations in accordance with the invention preferably arewater-soluble and maintain their activity throughout the shelf life of aproduct. EDTA is a preferred antioxidant in accordance with theinvention in this regard.

Formulations in accordance with the invention may include metal ionsthat are protein co-factors and that are necessary to form proteincoordination complexes, such as zinc necessary to form certain insulinsuspensions. Metal ions also can inhibit some processes that degradeproteins. However, metal ions also catalyze physical and chemicalprocesses that degrade proteins.

Magnesium ions (10-120 mM) can be used to inhibit isomerization ofaspartic acid to isoaspartic acid. Ca⁺² ions (up to 100 mM) can increasethe stability of human deoxyribonuclease. Mg⁺², Mn⁺², and Zn⁺², however,can destabilize rhDNase. Similarly, Ca⁺² and Sr⁺² can stabilize FactorVIII, it can be destabilized by Mg⁺², Mn⁺² and Zn⁺², Cu⁺² and Fe⁺², andits aggregation can be increased by Al+3 ions.

Embodiments of the heterodimeric antibody or fragment formulationsfurther comprise one or more preservatives. Preservatives are necessarywhen developing multi-dose parenteral formulations that involve morethan one extraction from the same container. Their primary function isto inhibit microbial growth and ensure product sterility throughout theshelf-life or term of use of the drug product. Commonly usedpreservatives include benzyl alcohol, phenol and m-cresol. Althoughpreservatives have a long history of use with small-moleculeparenterals, the development of protein formulations that includespreservatives can be challenging. Preservatives almost always have adestabilizing effect (aggregation) on proteins, and this has become amajor factor in limiting their use in multi-dose protein formulations.To date, most protein drugs have been formulated for single-use only.However, when multi-dose formulations are possible, they have the addedadvantage of enabling patient convenience, and increased marketability.A good example is that of human growth hormone (hGH) where thedevelopment of preserved formulations has led to commercialization ofmore convenient, multi-use injection pen presentations. At least foursuch pen devices containing preserved formulations of hGH are currentlyavailable on the market. Norditropin (liquid, Novo Nordisk), Nutropin AQ(liquid, Genentech) & Genotropin (lyophilized—dual chamber cartridge,Pharmacia & Upjohn) contain phenol while Somatrope (Eli Lilly) isformulated with m-cresol.

Several aspects need to be considered during the formulation anddevelopment of preserved dosage forms. The effective preservativeconcentration in the drug product must be optimized. This requirestesting a given preservative in the dosage form with concentrationranges that confer anti-microbial effectiveness without compromisingprotein stability.

As might be expected, development of liquid formulations containingpreservatives are more challenging than lyophilized formulations.Freeze-dried products can be lyophilized without the preservative andreconstituted with a preservative containing diluent at the time of use.This shortens the time for which a preservative is in contact with theprotein, significantly minimizing the associated stability risks. Withliquid formulations, preservative effectiveness and stability should bemaintained over the entire product shelf-life (about 18 to 24 months).An important point to note is that preservative effectiveness should bedemonstrated in the final formulation containing the active drug and allexcipient components.

Heterodimeric antibody or fragment formulations generally will bedesigned for specific routes and methods of administration, for specificadministration dosages and frequencies of administration, for specifictreatments of specific diseases, with ranges of bio-availability andpersistence, among other things. Formulations thus may be designed inaccordance with the invention for delivery by any suitable route,including but not limited to orally, aurally, opthalmically, rectally,and vaginally, and by parenteral routes, including intravenous andintraarterial injection, intramuscular injection, and subcutaneousinjection.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,crystal, or as a dehydrated or lyophilized powder. Such formulations maybe stored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration. The invention alsoprovides kits for producing a single-dose administration unit. The kitsof the invention may each contain both a first container having a driedprotein and a second container having an aqueous formulation. In certainembodiments of this invention, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are provided.

The therapeutically effective amount of an antigen bindingprotein-containing pharmaceutical composition to be employed willdepend, for example, upon the therapeutic context and objectives. Oneskilled in the art will appreciate that the appropriate dosage levelsfor treatment will vary depending, in part, upon the molecule delivered,the indication(s) for which the antigen binding protein is being used,the route of administration, and the size (body weight, body surface ororgan size) and/or condition (the age and general health) of thepatient. In certain embodiments, the clinicians may titer the dosage andmodify the route of administration to obtain the optimal therapeuticeffect.

Stability

The terms “stability” and “stable” as used herein in the context of acomposition comprising a heterodimeric antibody (or antigen bindingfragment thereof) refer to the resistance of the heterodimeric antibody(or antigen binding fragment thereof) in the composition to aggregation,degradation or fragmentation under given manufacture, preparation,transportation and/or storage conditions. Antibody formulationscomprising a high degree of stability demonstrate enhanced reliabilityand safety and, as such, are advantageous for clinical use.

Antibody stability in a composition is optionally assessed by examininga desired parameter of the antibody in the composition (e.g.,aggregation, degradation of heavy and/or light chains, chemicalmodification, etc.) over time. In this regard, a parameter is typicallyexamined at an initial time point (T0) and an assessment time point(T1), optionally while exposing the antibody or fragment thereof to anyof a number of environmental conditions, and compared. An initial timepoint can be, for instance, the time that the antibody or fragmentthereof is first formulated in a composition or first examined forquality (i.e., examined to determine whether the antibody compositionmeets regulatory or manufacturing specifications with respect toaggregation or degradation). An initial time point also can be the timeat which the antibody or antibody fragment is reformulated in acomposition (e.g., reformulated at a higher or lower concentrationcompared to an initial preparation). An assessment time point is, invarious embodiments, about 1 week (or about 2 weeks, or about 3 weeks,or about 4 weeks, or about 5 weeks, or about 6 weeks, or about 7 weeks,or about 8 weeks, or about 10 weeks, or about 3 months, or about 6months or about 1 year) after the initial time point. The desiredparameter (e.g., aggregation or degradation) of the antibody or fragmentthereof in the composition can be assessed under a variety of storageconditions, such as temperatures of −30° C., 4° C., 20° C. or 40° C.,shaking, pH, storage in different container materials (e.g., glassvials, pre-filled syringes, etc.), and the like.

Exemplary methods for determining the degree of aggregation, and/ortypes and/or sizes of aggregates present in a composition comprising theheterodimeric antibody include, but are not limited to, size exclusionchromatography (SEC), high performance size exclusion chromatography(HPSEC), static light scattering (SLS), Fourier Transform InfraredSpectroscopy (FTIR), circular dichroism (CD), urea-induced proteinunfolding techniques, intrinsic tryptophan fluorescence, differentialscanning calorimetry, and 1-anilino-8-naphthalenesulfonic acid (ANS)protein binding techniques. Size exclusion chromatography (SEC) may beperformed to separate molecules on the basis of their size, by passingthe molecules over a column packed with the appropriate resin, thelarger molecules (e.g. aggregates) will elute before smaller molecules(e.g. monomers). The molecules are generally detected by UV absorbanceat 280 nm and may be collected for further characterization. Highpressure liquid chromatographic columns are often utilized for SECanalysis (HP-SEC). Alternatively, analytical ultracentrifugation (AUC)may be utilized. AUC is an orthogonal technique which determines thesedimentation coefficients (reported in Svedberg. S) of macromoleculesin a liquid sample Like SEC, AUC is capable of separating and detectingantibody fragments/aggregates from monomers and is further able toprovide information on molecular mass. Antibody or antibody fragmentaggregation in a composition may also be characterized by particlecounter analysis using a coulter counter or by turbidity measurementsusing a turbidimeter. Turbidity is a measure of the amount by which theparticles in a solution scatter light and, thus, may be used as ageneral indicator of protein aggregation. In addition, non-reducingpolyacrylamide gel electrophoresis (PAGE) or capillary gelelectrophoresis (CGE) may be used to characterize the aggregation and/orfragmentation state of antibodies or antibody fragments in acomposition.

Exemplary methods for determining antibody degradation include, but arenot limited to, size-exclusion chromatography (SEC), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and capillaryelectrophoresis with SDS (CE-SDS) and reversed phase HPLC with in-lineMS detection.

In various embodiments, less than 5% of the heterodimeric antibody orantibody fragment described herein in the composition is in aggregateform under conditions of interest. For instance, less than 4%, or lessthan 3%, or less than 2%, or less than 1% of the heterodimeric antibodyor fragment thereof in the composition is in aggregate form afterstorage at −30° C., 4° C., 20° C. or 40° C. for a period of about 1 week(or about 2 weeks, or about 3 weeks, or about 4 weeks, or about 5 weeks,or about 6 weeks, or about 7 weeks, or about 8 weeks, or about 10 weeks,or about 3 months, or about 6 months or about 1 year). In someembodiments, less than 5% (or less than 4% or less than 3% or less than2% or less than 1% or less) of the heterodimeric antibody of antibodyfragment described herein in the composition is in aggregate form afterstorage for two weeks at about 4° C.

For example at least 85% (or at least 90%, or at least 91%, or at least92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%,or at least 97%, or at least 98%, or at least 99%) of antibody orfragment thereof in a composition optionally is present in non-aggregate(i.e., monomeric)form after storage at −30° C., 4° C., 20° C. or 40° C.for a period of about 1 week (or about 2 weeks, or about 3 weeks, orabout 4 weeks, or about 5 weeks, or about 6 weeks, or about 7 weeks, orabout 8 weeks, or about 10 weeks, or about 3 months, or about 6 monthsor about 1 year). In some embodiments, at least 85% (or at least 90%, orat least 91%, or at least 92%, or at least 93%, or at least 94%, or atleast 95%, or at least 96%, or at least 97%, or at least 98%, or atleast 99% or more) of the antibody or fragment thereof is present in thecomposition in non-aggregate form after two weeks of storage at about 4°C. In some embodiments, at least 99% of the antibody is present in thecomposition in non-aggregate form after storage for two weeks at about4° C. for two weeks and/or at least 95% of antibody present is in thecompositions is in non-aggregate form after storage for two weeks at 40°C.

In various embodiments, less than 5% of the heterodimeric antibody orantibody fragment described herein in the composition is degraded. Forinstance, less than 4%, or less than 3%, or less than 2%, or less than1% or less of the heterodimeric antibody or fragment thereof in thecomposition is degraded under conditions of interest. For example,optionally at least 85% (or at least 90%, or at least 91%, or at least92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%,or at least 97%, or at least 98%, or at least 99%) of the antibody orfragment is intact (i.e., not degraded) in a composition stored at about−30° C., about 4° C., about 20° C. or about 40° C. for a period of about1 week (or about 2 weeks, or about 3 weeks, or about 4 weeks, or about 5weeks, or about 6 weeks, or about 7 weeks, or about 8 weeks, or about 10weeks, or about 3 months, or about 6 months or about 1 year). In someaspects, at least 85% (or at least 90%, or at least 91%, or at least92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%,or at least 97%, or at least 98%, or at least 99% or more) of theantibody or fragment thereof is intact (i.e., non-degraded) afterstorage in a composition at about 4° C. for a period of two weeks. Insome embodiments, at least 99% of the antibody or fragment remainsintact when stored in a composition at about 4° C. for two weeks and/orat least 95% remains intact when stored in a composition at about 40° C.for two weeks.

Functional or activity stability of the heterodimeric antibody (orantigen binding fragment there) in a composition also is contemplatedherein. Assays for detecting and/or quantifying, e.g., antibody bindingto a target, sclerostin neutralization, and DKK-1 neutralization areknown in the art and are described herein in Examples 4-6. Optionally,the antibody or fragment thereof demonstrates about 50-100% activityunder conditions of interest compared to the activity of the antibody orfragment thereof at the initial time point. For example, the antibody orfragment thereof retains a level of activity of between about 60-90% or70-80% compared to the activity the initial time point. Accordingly,functional stability of the antibody or fragment thereof includesretention of activity of at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% and can include activity measurements greaterthan 100% such as 105%, 110%, 115%, 120%, 125% or 150% or more comparedto the activity at the initial time point.

Viscosity

In some embodiments, the viscosity of a composition comprising one ormore of the heterodimeric antibodies described herein is determined. Theterm “viscosity” as used herein refers to “absolute viscosity.” Absoluteviscosity, sometimes called dynamic or simple viscosity, is the productof kinematic viscosity and fluid density (Absolute Viscosity=KinematicViscosity×Density). The dimension of kinematic viscosity is L²/T where Lis a length and T is a time. Commonly, kinematic viscosity is expressedin centistokes (cSt). The SI unit of kinematic viscosity is mm²/s, whichis 1 cSt. Absolute viscosity is expressed in units of centipoise (cP).The SI unit of absolute viscosity is the millipascal-second (mPa-s),where 1 cP=1 mPa-s.

The viscosity of a composition can be measured hours (e.g., 1-23 hours),days (e.g., 1-10 days), weeks (e.g., 1-5 weeks), months (e.g., 1-12months), or years (e.g., 1-2 years, 1-3 years) after the addition of theantibody to the composition. Viscosity measurements may be made at astorage or administration temperature, e.g. 2-8° C. or 25° C. (roomtemperature). In some embodiments, absolute viscosity of the liquid orreconstituted liquid composition at the storage and/or administrationtemperature is 15 cP or less, or 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4cP or less. In some embodiments, absolute viscosity of the liquid orreconstituted liquid composition is 6 cP or less.

In some embodiments, the viscosity of the antibody composition ismeasured prior to and after the addition of heterodimeric antibody.Methods of measuring viscosity are well known in the art and include,for example, using a capillary viscometer, or a cone-plate rheometer.Any method may be used provided the same method is used to compare thetest and reference formulations.

Kits

A pharmaceutical composition comprising one or more heterodimericantibodies described herein may be placed within containers (e.g., vialsor syringes), along with packaging material that provides instructionsregarding the use of such pharmaceutical compositions. Generally, suchinstructions will include a tangible expression describing theheterodimeric antibody concentration, as well as within certainembodiments, relative amounts of excipient ingredients or diluents(e.g., water, saline or PBS) that may be necessary to reconstitute thepharmaceutical composition.

Additional Embodiments

Also contemplated are the following embodiments provided in thefollowing numbered paragraphs:

1. An isolated antibody heavy chain variable region comprising an aminoacid substitution within the framework region at AHo position 51 or 141,wherein the substitution introduces a positive- or negative-chargedamino acid into the heavy chain variable region framework at saidposition.

2. The isolated antibody heavy chain variable region of paragraph 1,wherein AHo position 51 or AHo position 141 is substituted for apositive charged amino acid.

3. The isolated antibody heavy chain variable region of paragraph 2,further comprising a substitution at AHo position 46 for a positivecharged amino acid.

4. The isolated antibody heavy chain variable region of paragraph 2 orparagraph 3, wherein AHo position 51 is substituted for a positivecharged amino acid.

5. The isolated antibody heavy chain variable region of paragraph 1, 2,or 3, wherein AHo position 141 is substituted for a positive chargeamino acid.

6. The isolated antibody heavy chain variable region of paragraph 3,wherein AHo position 46 and AHo position 141 are substituted for apositive charged amino acid.

7. The isolated antibody heavy chain of any of paragraphs 2-6, whereinthe positive charged amino acid is lysine.

8. The isolated antibody heavy chain variable region of paragraph 1,wherein AHo position 51 or AHo position 141 is substituted for anegative charged amino acid.

9. The isolated antibody heavy chain variable region of paragraph 8,further comprising a substitution at AHo position 46 for a negativecharged amino acid.

10. The isolated antibody heavy chain variable region of paragraph 8 orparagraph 9, wherein AHo position 51 is substituted for a negativecharged amino acid.

11. The isolated antibody heavy chain variable region of paragraph 8, 9,or 10, wherein AHo position 141 is substituted for a negative chargedamino acid.

12. The isolated antibody heavy chain variable region of paragraph 9,wherein AHo position 46 and AHo position 141 are substituted for anegative charged amino acid.

13. The isolated antibody heavy chain of any of paragraphs 8-12, whereinthe negative charged amino acid is aspartic acid.

14. The isolated antibody heavy chain variable region of any ofparagraphs 1-13, further comprising a heavy chain CH1 region.

15. The isolated antibody heavy chain variable region of paragraph 14,wherein the CH1 region comprises one or more amino acid additions,deletions, or substitutions.

16. The isolated antibody heavy chain variable region of paragraph 15,wherein an amino acid is substituted to introduce a positive- ornegative-charged amino acid into the CH1 region.

17. The isolated antibody heavy chain variable region of paragraph 16,wherein the positive- or negative-charged amino acid is introduced at EUposition S183.

18. The isolated antibody heavy chain variable region of paragraph 17,wherein EU position S183 is substituted for a positive-charged aminoacid.

19. The isolated antibody heavy chain variable region of paragraph 18,wherein the substitution is S183K.

20. The isolated antibody heavy chain variable region of paragraph 17,wherein EU position S183 is substituted for a negative-charged aminoacid.

21. The isolated antibody heavy chain variable region of paragraph 20,wherein the substitution is S183D.

22. An antibody heavy chain comprising the isolated antibody heavy chainvariable region of any of paragraphs 1-21.

23. The antibody heavy chain of paragraph 22, wherein the heavy chaincomprises a CH3 region comprising one or more amino acid substitutionsdisfavoring homodimerization.

24. The antibody heavy chain of paragraph 23, wherein a negative chargedamino acid in the CH3 region is substituted with a positive chargedamino acid.

25. The antibody heavy chain of paragraph 24, wherein the negativecharged amino acid is EU position D399, E356, or E357.

26. The antibody heavy chain of paragraph 25, wherein the positivecharged amino acid is lysine.

27. The antibody heavy chain of paragraph 26, wherein the CH3 regioncomprises D399K and E356K substitutions.

28. The antibody heavy chain of paragraph 23, wherein a positive chargedamino acid in the CH3 region is substituted with a negative chargedamino acid.

29. The antibody heavy chain of paragraph 28, wherein the positivecharged amino acid is EU position K370, K392, or K409.

30. The antibody heavy chain of paragraph 29, wherein the negativecharged amino acid is aspartic acid.

31. The antibody heavy chain of paragraph 30, wherein the CH3 regioncomprises K392D and K409D substitutions.

32. An antibody comprising the antibody heavy chain of any of paragraphs24-27 and the antibody heavy chain of any of paragraphs 28-31.

33. The antibody heavy chain of any of paragraphs 22-31, wherein theheavy chain comprises a CH2 region comprising one or more amino acidsubstitutions altering an Fc effector function.

34. An antibody kappa light chain variable region comprising an aminoacid substitution within the framework region at AHo position 51 or 141,wherein the substitution introduces a positive- or negative-chargedamino acid into the kappa light chain variable region framework at saidposition.

35. The antibody kappa light chain variable region of paragraph 34,wherein AHo position 51 or AHo position 141 is substituted for apositive charged amino acid.

36. The antibody kappa light chain variable region of paragraph 35,further comprising a substitution at AHo position 46 for a positivecharged amino acid.

37. The antibody kappa light chain variable region of paragraph 35 or36, wherein AHo position 51 is substituted for a positive charged aminoacid.

38. The antibody kappa light chain variable region of paragraph 35, 36,or 37, wherein AHo position 141 is substituted for a positive chargedamino acid.

39. The antibody light chain of any of paragraphs 35-38, wherein thepositive charged amino acid is lysine.

40. The antibody kappa light chain variable region of paragraph 34,wherein AHo position 51 or AHo position 141 is substituted for anegative charged amino acid.

41. The antibody kappa light chain variable region of paragraph 40,further comprising at substitution at AHo position 46 for a negativecharged amino acid.

42. The antibody kappa light chain variable region of paragraph 40 or41, wherein AHo position 51 is substituted for a negative charged aminoacid.

43. The antibody kappa light chain variable region of paragraph 40, 41,or 42, wherein AHo position 141 is substituted for a negative chargedamino acid.

44. The antibody light chain of any of paragraphs 40-43, wherein thepositive charged amino acid is lysine.

45. The antibody kappa light chain variable region of any of paragraphs34-44, further comprising a kappa light chain constant region.

46. The antibody kappa light chain variable region of paragraph 45,wherein the kappa light chain constant region comprises one or moreamino acid additions, deletion, or substitutions.

47. The isolated antibody kappa chain variable region of paragraph 46,wherein an amino acid is substituted to introduce a positive- ornegative-charged amino acid into the kappa light chain constant region.

48. The isolated antibody kappa chain variable region of paragraph 47,wherein the positive- or negative-charged amino acid is introduced at Euposition S 176.

49. The isolated antibody kappa chain variable region of paragraph 48,wherein Eu position S176 is substituted for a positive charged aminoacid.

50. The isolated antibody kappa chain variable region of paragraph 49,wherein the substitution is S176K.

51. The isolated antibody kappa chain variable region of paragraph 48,wherein Eu position S176 is substituted for a negative charged aminoacid.

52. The isolated antibody kappa chain variable region of paragraph 51,wherein the substitution is S176D.

53. An antibody lambda light chain variable region comprising an aminoacid substitution within the framework region at AHo position 51 or 141,wherein the substitution introduces a positive- or negative-chargedamino acid into the lambda light chain variable region framework at saidposition.

54. The antibody lambda light chain variable region of paragraph 53,further comprising a lambda light chain constant region.

55. The antibody lambda light chain variable region of paragraph 54,wherein the lambda light chain constant region comprises one or moreamino acid additions, deletion, or substitutions.

56. The isolated antibody lambda chain variable region of paragraph 55,wherein an amino acid is substituted to introduce a positive- ornegative-charged amino acid into the lambda light chain constant region.

57. The isolated antibody lambda chain variable region of paragraph 56,wherein the positive- or negative-charged amino acid is introduced atKabat position S 176.

58. The isolated antibody lambda chain variable region of paragraph 57,wherein S176 is substituted for a positive charged amino acid.

59. The isolated antibody lambda chain variable region of paragraph 58,wherein the substitution is S176K.

60. The isolated antibody lambda chain variable region of paragraph 57,wherein S176 is substituted for a negative charged amino acid.

61. The isolated antibody lambda chain variable region of paragraph 60,wherein the substitution is S176D or S176E.

62. An isolated nucleic acid encoding the antibody heavy chain variableregion of any of paragraphs 1-21, the antibody heavy chain of any ofparagraphs 22-31, the antibody kappa light chain variable region of anyof paragraphs 34-52, or the antibody light chain variable region of anyof paragraphs 53-61.

63. An expression vector comprising the isolated nucleic acid ofparagraph 62 operably linked to a promoter.

64. A recombinant host cell comprising the isolated nucleic acid ofparagraph 62.

65. A recombinant host cell comprising the expression vector ofparagraph 63.

66. An antigen binding protein comprising the isolated heavy chainvariable region on any of paragraphs 1-21 and the isolated light chainvariable region of any of paragraphs 22-31 or 34-52.

67. The antigen binding protein of paragraph 66, wherein the antigenbinding protein is an antibody comprising two heavy chains and two lightchains.

68. The antigen binding protein of paragraph 67, wherein the antibody isa bi-specific antibody.

69. A pharmaceutical composition comprising the antigen binding proteinof any of paragraphs 66-68.

EXAMPLES Example 1 Generation of Linkerbodies

The following Example describes the generation of heterodimericantibodies (with an IgG1 backbone) wherein the light and heavy chains of(a) an anti-sclerostin antibody and (b) an anti-DKK1 antibody arecovalently linked using a G4S linker to form a single polypeptide chainthat binds sclerostin and a single polypeptide chain that binds DKK1(“linkerbodies”). The polypeptide chains or half antibodies against theDKK1 and sclerostin targets are then assembled as a bispecific antibodythrough charge pair substitutions at the CH3 domain (i.e., one heavychain contained K392D and K409D substitutions and the other containedE356K and D399K substitutions). The charge pair substitutions employ theelectrostatic steering mechanism described herein, whereby heterodimerformation (DKK1 Ab—sclerostin Ab) is encouraged by attraction betweennegatively- and positively-charged residues in the CH3 regions andhomodimers (two DKK1 Ab arms or two aclerostin Ab arms) are discourageddue to repulsion between amino acids having the same charge atcorresponding regions of the CH3-CH3 interface. Charge pairsubstitutions were also introduced at the CH1-CL domain interface inorder to reduce the level of aggregation (S183K/E in the heavy chain andS176E/K in the light chain). Further, some of the linkerbodies also hadengineered inter-domain disulfide bonds at the VL-VH interface forfurther stabilization of heavy-light chain interactions. This wasachieved by substituting G44 (Kabat) in the heavy chain and G100 (Kabat)in the light chain to cysteine residues.

The methods described above resulted in a high level of aggregation asdetermined by Size Exclusion Chromatography (SEC). In order to reducethe level of aggregation, a G4S linker was either added or replacedbetween the light and heavy chains with a charge pair substitution inthe heavy and light chain constant regions (CH1-CL1). It wascontemplated that the aggregation level would be reduced as a result ofadding the charge pair substitution to the linkerbody constructs.

The linkerbody design in combination with charge pair substitutions inthe CH1-CL interface still resulted in unwanted aggregation issues inseveral constructs.

Example 2 Generation of Heterodimeric Antibody without Linkers

The following Example describes the generation of a heterodimericantibody having no linkers but, instead, comprising charge pairsubstitution in both the CH/CL interface and the CH3/CH3 interface ofthe parent antibodies. The resulting heterodimeric antibody comprisingcharged substitutions in the CH/CL and CH3/CH3 interfaces is referencedherein as heterodimeric antibody version 1 or heteroIg-v1.

Briefly, the following substitutions were introduced into DKK1 antibody6.37.5: K392D (EU) and K409D (EU) substitutions in the CH3 domain, S183K(EU) substitution in the CH1 domain, and S176E (EU) substitution in theCL domain. The following substitutions were introduced into sclerostinantibody 27H6: E356K (EU) and D399K (EU) substitutions in the CH3domain, S183E (EU) substitution in the CH1 domain, and S176K (EU)substitution in the CL domain. IgG1 scaffold was used in theheterodimeric version 1design.

Various input DNA ratios for the two different antibodies were used tomaximize the IgG production and minimize the aggregation level (HighMolecular Weight species). Using equal amount of DNA for both antibodiesled to minimal aggregation level.

In order to assess the hetero Ig bispecific antibody formation, thematerial was purified and subjected to mass spectrometry analysis. NRmass analysis confirmed that the antibody product had two differentlight chains and two different heavy chains. To confirm the specificlight-heavy chain pairing, the Fab fragments were generated throughproteolysis (Pierce Fab Micro Preparation Kit). The mass analysis showedonly two species, one corresponding to the DKK1 antibody heavy and lightchain pairing and the other corresponding to that of a sclerostinantibody. The mass analysis confirmed the presence of heterodimericantibodies having the correct pairing of light and heavy chains in boththe arms of sclerostin/DKK1 heterodimeric antibody.

The mass analysis indicated the presence of a single species of antibodyin the purified sample, with the observed mass matching the calculatedmass of the heterodimeric antibody. The resulting heterodimeric antibodyhad two light chains and two heavy chains, with the heavy chain of theanti-sclerostin portion of the heterodimeric antibody having a S183E(EU) substitution in the CH1 domain and the light chain of theanti-sclerostin portion having a S176K (EU) substitution in the CLdomain. The heavy chain of the anti-DKK1 portion of the heterodimericantibody had a S183K (EU) substitution the CH1 domain and the lightchain of the DKK1 portion had a S176E (EU) substitution in the CLdomain. The CH3 domain of the anti-sclerostin portion of theheterodimeric antibody had E356K (EU) and D399K (EU) substitutions andthe CH3 domain of the anti-DKK1 portion had substitutions at K392D (EU)and K409D (EU).

The ability of the generated heterodimeric antibodies to activatecanonical Wnt signaling in the presence of sclerostin and/or DKK1 wasevaluated in an independent osteoblast Wnt activation assay where cellsare induced to differentiate and secrete factors that activate Wntsignaling in an autocrine fashion. In the assay, MC3T3-E1 cells weretransfected with a Super-TOPFlash reporter construct, and stable celllines were selected and evaluated. MC3T3E1/TetONWnt1/Luciferase is amouse osteblast cell line engineered with a T-Cell factor responseluciferase construct, Tet Repressor construct and a doxycyclineinducible Wnt-1 construct using lentiviral transduction. In the presenceof doxycycline, the MC3T3E1/TetONWnt1/Luc cells express Wnt-1 and inducesignal transduction via the binding of Wnt-1 to cell surface LRP5/6 andFrizzled receptors resulting in the expression of luciferase. WhenMC3T3E1/TetONWnt1/Luc#5 cell are incubated in the presence of sclerostinand/or DKK1 Wnt signaling is inhibited by these proteins via the Lrp5/6beta propeller 1 motif. The bioassay measures the dose dependentstimulatory effect in the cell-based reporter assay of the heterodimericantibody and parental antibodies treated with a fixed concentration ofsclerostin and/or DKK1.

Clone C10 demonstrated decreased reporter activity following incubationwith either purified sclerostin or DKK1 proteins due to inhibition ofWnt pathway activation. Cells were cultured in Expansion Medium(Alpha-MEM medium containing 10% FBS, 1× Pen-Strep-Glu and 1.0 ug/ml ofpuromycin). When the cells reached 80% confluence, the medium wasswitched to Differentiation Medium “DM” (Expansion Medium, 50 ug/mlascorbic acid and 10 mM beta-glycerophosphate) for 4 days. Followingdifferentiation, this cell line produced an endogenous protein(s) thattriggers canonical Wnt activation in an autocrine manner. Media wasaspirated and 1000s of fresh DM containing various concentrations ofmonospecific or heterodimeric antibodies (pre-incubated for 4 hours withDKK1 and/or sclerostin for 45-60 min at 37° C.) was added to the wellsfor 24 hours. Luciferase activity was measured following manufacturer'sinstructions (Promega's Luciferase Assay System, Cat No: E4530). Variousrat and human bispecific antibodies tested were capable ofdose-dependently activating the osteoblast canonical Wnt pathway in thepresence of both sclerostin and DKK1, further demonstrating that theantibodies can simultaneously neutralize the Wnt inhibitory function ofboth soluble proteins.

Results indicated that both the heterodimeric antibody producedaccording to the methods described in this Example and the linkerbody(which was used as a positive control) had very similar activity,further confirming correct pairing of the light and heavy chains.

Example 3 Generation of Heterodimeric Antibodies Having Substitutions inCH3 Domain(s), CH/CL Domain(s) and VH/VL Domain(s)

The following Example describes the generation of heterodimericantibodies having one or more substitutions in each of the CH3, CH/CLand VH/VL domains to further favor correct pairing of the light andheavy chains. The heterodimeric antibodies are based on either Ab-5 andAb-23 for the sclerostin portion and antibodies 6.147 and 6.37.5 for theDKK1 portion. An IgG2 class constant domain was used here in order toprevent ADCC and discourage effector functions. The resultingheterodimeric antibody comprising charged substitutions in the VH/VL,CH/CL and CH3/CH3 interfaces is referenced herein as heterodimericantibody version 2 or heterolg-v2.

When co-expressing two different antibodies inside one cell, fourdifferent chains (HCl, LC1, HC2, LC2) are transcribed and translated.HCs can form either homodimer (HC1-HC1) or heterodimer (HC1-HC2); LCscan randomly assemble with two different HCs. In all, ten differentcombinations can occur [Paul Carter J Immunological Methods 248 (2001)7-15]. Undesired heavy chain homodimers can be minimized by engineeringthe C_(H)3 region to only form a heterodimer. This Example demonstratesthat undesirable LC/HC pairings can be eliminated by engineering theinterface of the LC/HC to enforce the correct pairing of LCs with theircognate HCs. An electrostatic-steering mechanism was applied to directthe pairing and assembly of LC/HC; opposite polarity attracts desiredcomplex subunits while the same polarity of homodimer subunits isrepulsive.

Several criteria were applied when selecting the pairs of residues alongthe heavy chain and light chain interface for replacement by chargedresidues with opposing polarity, e.g., Asp or Lys, to control thecorrect pairing of LC with its cognate HC: 1) All positions are locatedin close proximity within both the VL/VH and CL/C_(H)1 interfaces; 2)All positions are buried and are well conserved among most, if not all,of different antibody families; 3) All positions have minimal impact onexpression and antigen binding; and 4) The introduction of chargedresidues does not interfere with the binding of chaperone BiP to theC_(H)1 region in the process of antibody folding and assembly.

The selected residues at the VL/VH and CK/C_(H)1 interfaces forengineering are listed in Table 2. In the variable regions, predominantAHo position 46 (Q39 Kabat), AHo position 51 (Kabat G44), and AHoposition 141 (Kabat Q105) in VH are in close proximity to AHo position46 (Kabat Q38), AHo position 141 (Kabat Q100), and AHo position 51(Kabat A43) in VL, respectively. In the constant regions, A141 (EU),P171 (EU), and S183 (EU) in C_(H)1 region contact residues F116 (EU),S162 (EU), and S176 (EU) in Ck respectively, but K147 (EU) in C_(H)1 caninteract with either Q124 (EU), S131 (EU), or T180 (EU) in Ck region.

TABLE 2 The amino acid residues located at the VH/VL and CH1/Cκinterfaces were selected for the introduction of charge pair residues.Germline residues of VH and VL are numbered by different numberingsystems, the bolded residues are the dominant ones. The contact residuesin VH/VL of most of antibodies are arrayed in the same row. Residues ofhuman IgG1 CH1 domain contacting the residues in Cκ region are alsobolded and laid in the same row. FW: Framework. VH VL IMGT # Kabat # Eu# AHo # FW Residue contact IMGT # Kabat # Eu # AHo # FW Residue 44 39 3946 2 Q/R/T

44 38 38 46 2 Q/G/H/L 49 44 44 51 2 G/R/A

* 100  100  141  4 Q/G/P * 105  109  141  4 Q/K/R/S

49 43 43 51 2 A/G/S/P C_(H)1 Cκ IMGT # Kabat # Eu # AHo # Ref # Residuecontact IMGT # Kabat # Eu # AHo # Ref # Residue 20 139 141 * 176 A

 5 116 116 * 158 F 82 175 171 * 212 P

81 162 162 * 211 S 86 188 183 * 230 S

86 176 176 * 230 S 26 145 147 * 182 K

13 124 124 * 166 Q 26 145 147 * 182 K

20 131 131 * 176 S 26 145 147 * 182 K

90 180 180 * 234 T

Wnt1-driven osteoblast reporter analysis of the heterodimeric antibodiesshowed robust activation of canonical Wnt signaling in the presence ofboth DKK1 and Sclerostin whereas control DKK1 antibodies (6.37.5, 6.147)and control Sclerostin antibody (Ab-23) only partially reversedinhibition.

The engineered heterodimeric antibodies were capable of neutralizingDKK1 and blocking recombinant Wnt3a induced TCF/LEF luciferase activityas seen in an independent cell based assay. In this assay, Wnt signalingwas induced in an MC3T3 E1/STF-luciferase stable cell line with 100ng/ml of recombinant Wnt3a protein (R&D Systems) for 30 minutes at 4° C.Cells were subsequently treated with human DKK1 protein at 0.15 ug/mlthat was pre-mixed with control PBS or a two-fold serial dilution of theheterodimeric antibodies starting at 426.7 nM. The luciferase signal wasdetermined after 24 hours as described above and the data were plottedby using PRISM software. Results indicated that the heterodimericantibodies with variable regions targeting both the loop 2 region ofsclerostin (i.e., amino acids 86-111 of SEQ ID NO: 1) and DKK1 werecapable of neutralizing Sclerostin and DKK1 and increased reporteractivity driven by Wnt3a.

Example 4 Functional Analysis of Parental and Heterodimeric Antibodies

Functional analysis of anti-Sclerostin parental antibodies directedagainst the loop 2 region of sclerostin (Ab23, Ab5, 20C3) and againstnon-loop 2 epitopes (27H6, Ab13, Ab-D, Ab-3) revealed a unique mechanismof action for each group of antibodies. A competition alpha screenbinding assay was conducted to measure the effects of increasingconcentrations of parental sclerostin antibodies on the interactionbetween his-tagged LRP6 and biotin-labeled human sclerostin. Thisanalysis revealed that where anti-sclerostin antibodies that bound theloop 2 region of sclerostin potently inhibited the interaction betweensclerostin and LRP6, antibodies directed against non-loop2 regions ofsclerostin increased the interaction between these proteins.Furthermore, the engineered heterodimeric antibodies showed a similarphenomenon where heterodimeric antibodies directed against non-loop 2sclerostin epitopes (i.e., 27H6-6.37.5, 6.147-27H6) promoted increasedbinding of LRP6 to sclerostin unlike the heterodimeric antibodiesdirected again the loop 2 region of sclerostin. All heterodimericantibodies were found to compete with human DKK1 for binding to LRP6.

To understand the impact of this phenomenon on canonical Wnt pathwayactivation, parental antibodies and heterodimeric antibodies werestudied in TCF-reporter cell based assays using osteoblasts and 293cells. Cells were stimulated with either Wnt1 which binds to LRP6 betapropeller 1 motif or Wnt3a which binds to the LRP6 beta propeller 3motif. Canonical Wnt pathway activation was measured by increasedluciferase activity. In the assays, sclerostin and/or DKK1 were added toinhibit reporter activity and the neutralizing activity of differentanti-sclerostin or anti-DKK1 antibodies was studied. Whereas sclerostininhibited Wnt3a-driven activation of the TCF reporter, ananti-sclerostin non-loop 2 region binding antibody (27H6) but notcontrol loop 2 binding antibodies (Ab5 and Ab23) potentiated Wnt3asignaling. Similar Wnt3a potentiation results were obtained withheterodimeric antibodies containing anti-sclerostin variable regionsagainst non-loop 2 regions of sclerostin. In contrast, Wnt1 activity wasrestored by sclerostin inhibition with either antibody as shown byincreased luciferase activity that did not exceed that of untreatedcontrols. These effects were dependent on the presence of sclerostinsince no potentiation was observed in the absence of sclerostin with theheterodimeric antibodies directed to the non-loop 2 regions ofsclerostin. A similar analysis of other heterodimeric antibodies showedthat heterodimeric antibodies comprising an anti-sclerostin portion thatbound loop 2 of sclerostin failed to potentiate Wnt3a, whereasheterodimeric antibodies containing variable regions that boundnon-loop2 regions of sclerostin did potentiate Wnt3a signaling. Inaddition, heterodimeric antibodies comprising variable regions thatbound non-loop 2 regions of sclerostin potentiated Wnt3a signaling inthe presence of DKK1, whereas monospecific non-loop 2 heterodimericantibody potentiation is inhibited in the presence of DKK1. Allcanonical Wnt activation was inhibited in the presence of DKK1 aloneconsistent with the recent reports showing that the C-terminal region ofDKK1 binds to the beta propeller 3 motif of LRP6 and blocks theinteraction of Wnt3a class proteins with this region (Cheng et al,Nature Structural Mol Biol, 2011; Anh Dev Cell 2011). These data suggestthat antibody-ligand complexes bound to the beta propeller domain 1 viasclerostin may impact the conformation of the receptor in such a manneras to increase Wnt3a/beta propeller 3 activity. A similarWnt3a-dependent potentiation phenomenon by anti-LRP6 beta propeller1-binding antibodies has previously been reported where these antibodiesincreased mitogenicity in cancer cell lines and growth of tumorxenografts (Ettenberg et al, PNAS 2010).

Wnt3a-potentiation by non-loop 2 binding anti-sclerostin antibodies wasalso observed in non-osteoblastic cells. This result raised thepossibility that sclerostin/non-loop 2 binding antibody complexes couldactivate Wnt signaling in any cell type expressing LRP6 provided Wnt3class proteins or beta propeller 3 binding proteins are present and DKK1is absent. In the case of a heterodimeric antibody, potentiation mayoccur in the presence of DKK1. Based on these observations and tomitigate risk of mitogenicity in non-osteoblastic cells, newheterodimeric antibodies were engineered with anti-sclerostin variableregions directed against the loop 2 region which were shown to robustlyincrease reporter activity in the Wnt1-osteoblast TCF reporter assay.

In addition, parental anti-sclerostin antibodies that bind non-loop 2regions of sclerostin do not disrupt the interaction of sclerostin withits cognate LRP receptors and preserve either LRP6 interactions or LRP4interactions. Although parental sclerostin antibodies against the loop 2region of sclerostin and heterodimeric antibodies inhibited theinteraction between LRP6 and sclerostin, they failed to inhibit theinteraction between sclerostin and LRP4 in co-immunoprecipitationexperiments. In contrast, parental antibodies or heterodimericantibodies containing anti-non-loop 2 variable domains all decreasedbinding between LRP4 and Sclerostin (19D11, 27H6, L8 and N22). Newheterodimeric antibodies were engineered in an IgG2 backbone.

Example 5 Heterodimeric Antibodies Demonstrated Activity In Vivo

The following Example demonstrates that heterodimeric antibodiesgenerated according to the methods described in Examples 2 and 3increased bone mineral density in an animal model of low bone mineraldensity.

Male 10 week old B6D2F1 mice were used in this study. At the beginningof the study, animals were divided into 5 groups (n=9/group), balancedby both body weight and bone mineral density (BMD) at the femur-tibiaregion by in vivo DXA. Mice were subcutaneously injected with eithervehicle (proline) or sclerostin-Ab (Scl-Ab), or DKK1-Ab or combinationof Scl-Ab and DKK1-Ab (combination) or heterodimeric antibody (heteroIg) twice per week for 3 weeks. The antibodies were dosed at 12.5 mg/ml.Animals were scanned weekly by in vivo DXA to monitor the bone anabolicactivity of the drug treatments at lumbar vertebral and femur-tibiaregions. The mice were subsequently euthanized at the end of study.Femurs were collected for ex vivo densitometry by μCT and bone strengthanalysis.

FIG. 1 illustrates the in vivo study design for the followingheterodimeric antibodies: (1) Ab23-Ab6.147 v2, (2) Ab5-Ab6.37.5 v1, and(3) Ab5-Ab6.147 v1. Ab-5 is used as mono-therapy control and DVD Ig6.147-Ab23 (described in International Publication No. WO 2012/118903)is used as bispecific antibody control in this study. The DVD Ig wasdosed slightly higher (17 mg/kg) due to the higher molecular weight thanthe monotherapy control and heterodimeric antibodies. It must be notedhere that the heterodimeric antibody format is monovalent against eachtarget, whereas the DVD is bivalent against each target. Therefore,although in terms of molarity, the dosing level makes DVD andheterodimeric antibody (hetero Ig) equivalent, they are different interms of number of binding sites (paratopes).

FIG. 2 compares percentage of bone mass density (BMD) increase in lumbarvertebrae and leg in mice between monospecific Ig (Ab5), bispecific DVD(6.147-Ab23) and heterodimeric antibodies 1, 2, & 3 described above atweek 3.

The data shown in FIG. 2 clearly demonstrate heterodimeric antibodiesAb5-6.37.5 v1 (heterodimeric antibody 2—“heteroIg2”) and 785-6.147 v1(heterodimeric antibody 3—“heteroIg3”) increased the bone mass density.However, the heterodimeric antibody Ab23-6.147 v2 (heterodimericantibody 1—“heteroIg1”) which has extra charge pair substitution in thevariable domain interface (VH/VL) did not increase the BMD. It wasexpected that adding additional charge pair substitution in the variabledomain would help achieve specific pairing of the light and heavy chains(e.g., Ab5 light pairing with Ab5 heavy chain). But, it appears that inthis case the additional charge pair substitution in the variable domainled to poor stability of the heterodimeric antibody. The PK study thatfollowed this PD study indeed demonstrated DKK1 arm of the heterodimericantibody Ab23-6.147 having poor in vivo stability.

Example 6 Pharmacokinetic Data

Four different pharmacokinetic assays (i.e., sclerostin/DKK1 Assay;sclerostin/Fc Assay; DKK1/sclerostin Assay; and FC/FC bridging ELISAAssay) were performed in order to capture and detect heterodimericantibodies in serum samples.

Sclerostin/DKK1 Assay: Briefly, half area plates were coated with 1μg/ml of human sclerostin in 1×PBS and incubated overnight at 4° C. Theplates were blocked for at least 1 h by I-Block buffer. Standards(Stds)) and quality control samples (QCs) were prepared in Rat serumsamples. Standards, QCs, and serum samples were diluted 1:30 in buffer(1×PBS, 1M NaCl, 0.5% tween 20, and 10 mg/ml BSA). The diluted Stds,QCs, and samples were then loaded into ELISA plate and incubated for 90min. Then the ELISA plate was washed and 200 ng/ml of biotinylated humanDKK1 in the buffer was added and incubated for 90 minutes. The plate waswashed and 200 ng/ml Streptavidin-HRP conjugated added and incubated for30 minutes. After washing the plate, TMB substrate was added. Thereaction was stopped after 15 minutes by addition of 1 M of sulfuricacid. The plate was then read by a SpectraMax plate reader.

Sclerostin/FC Assay:

Half area plates were coated with 1 μg/ml of human sclerostin in 1×PBSand incubated overnight at 4° C. The plates were blocked for at least 1hour by I-Block buffer. Standards (Stds)) and quality control samples(QCs) were prepared in Rat serum samples. Standards, QCs, and serumsamples were diluted 1:30 in buffer (1×PBS, 1M NaCl, 0.5% tween 20, and10 mg/ml BSA). The diluted Stds, QCs, and samples were then loaded intoELISA plate and incubated for 90 minutes. Then the ELISA plate waswashed and 10 ng/ml of HRP conjugated anti-human Fc antibody, Mab 1.35.1was added and incubated for 90 minutes. After washing the plate, TMBsubstrate was added. The reaction was stopped after 15 minutes byaddition of 1 M of sulfuric acid. The plate was then read by aSpectraMax plate reader.

DKK1/FC Assay:

Half area plates were coated with 1 μg/ml of human DKK1 in 1×PBS andincubated overnight at 4° C. The plates were blocked for at least 1 hourby I-Block buffer. Standards (Stds)) and quality control samples (QCs)were prepared in Rat serum samples. Standards, QCs, and serum sampleswere diluted 1:30 in buffer (1×PBS, 1M NaCl, 0.5% tween 20, and 10 mg/mlBSA). The diluted Stds, QCs, and samples were then loaded into ELISAplate and incubated for 90 min. Then the ELISA plate was washed and 10ng/ml of HRP conjugated anti-hu Fc antibody, Mab 1.35.1 was added andincubated for 90 minutes. After washing the plate, TMB substrate wasadded. The reaction was stopped after 15 minutes by addition of 1 M ofsulfuric acid. The plate was then read by a SpectraMax plate reader.

FC/FC Bridging ELISA Assay:

Briefly, half area plates were coated with 0.2 μg/ml of anti-human Fcantibody, Mab 1.35.1 in 1×PBS and incubated overnight at 4° C. Theplates were blocked for at least 1 hour by I-Block buffer. Standards(Stds) and quality control samples (QCs) were prepared in rat serumsamples. Standards, QCs, and serum samples were diluted 1:30 in buffer(1×PBS, 1M NaCl, 0.5% tween 20, and 10 mg/ml BSA). Then, the dilutedStds, QCs, and samples were loaded into ELISA plate and incubated for 90min. Then the ELISA plate was washed and 50 ng/ml of HRP conjugatedanti-human Fc antibody, Mab 1.35.1 was added and incubated for 30 min.After washing the plate, TMB substrate was added. The reaction wasstopped after 10 minutes by addition of 1 M of sulfuric acid. The platewas then read by a SpectraMax plate reader.

FIG. 3 shows the pharmacokinetic (PK) profiles of the foursclerostin-DKK1 heterodimeric antibodies tested (i.e., Ab23-6.37.5 v1,Ab5-6.37.5 v1, Ab5-6.147 v2 and Ab23-6.147 v2). Results indicated thatheterodimeric antibodies comprising a charged amino acid substitutionsin the VH/VL domains negatively impacted the stability as the PKprofiles based on the sclerostin/DKK1 & DKK1/Fc assays deviated fromthat of the sclerostin/Fc & Fc/Fc assays.

Example 7 Controlling the Correct Pairing of Light Chain with itsCognate Heavy Chain During the Production of Heterodimeric Antibodies byElectrostatic Steering Mechanism

When co-expressing two different antibodies inside one cell, fourdifferent chains (HC1, LC, HC2, LC2) are transcribed and translated. HCscan form either homodimer or heterodimer; LCs can randomly assemble withtwo different HCs. Ten different combinations can occur [Paul Carter JImmunological Methods 248 (2001) 7-15]. The undesired side productsderived from heavy chain homodimer can be minimized by engineering theCH3 region to only form a heterodimer. This Example demonstrates thatundesirable LC/HC pairings can be eliminated by engineering theinterface of the LC/HC to enforce the correct pairing of LCs with theircognate HCs. An electrostatic-steering mechanism was applied to directthe pairing and assembly of LC/HC, as the opposite polarity isattractive while the same polarity is repulsive.

Several criteria were applied when selecting the pairs of residues alongthe heavy chain and light chain interface that were replaced by chargedresidues with opposing polarity, e.g., Asp or Lys, to control thecorrect pairing of LC with its cognate HC: 1) All positions are locatedin close proximity within both the VL/VH and CL/CH1 interfaces; 2) Allpositions are buried and are well conserved among most, if not all, ofdifferent antibody families; 3) All positions have minimal impact onexpression and antigen binding; and 4) The introduction of chargedresidues does not interfere with the binding of chaperone BiP to the CH1region in the process of antibody folding and assembly.

The selected residues at the VL/VH and Cκ/CH1 interfaces for engineeringHer2/EGFR heterodimeric antibodies are listed in Table 2. In thevariable regions, predominant Q39 (Kabat numbering; AHo position 46),G44 (AHo position 51), and Q105 (AHo position 141) in VH are in closeproximity to Q38 (Kabat numbering; AHo position 46), Q100 (AHo position141), and A43 (AHo position 51) in VL, respectively. In the constantregions, A141 (Eu numbering), P171, and S183 in CH1 region contactresidues F116 (Eu numbering), S162, and S176 in Ck respectively, butK147 (Eu numbering) in CH1 can interact with either Q124, S131, or T180(Eu numbering) in Ck region.

A proof of concept heterodimeric IgG was constructed using v-genes froman anti-EGFR antibody and an anti-HER2 antibody. FIG. 4 shows differentconfigurations for making heterodimeric antibody variants. One Fc chainhas ADCC-enhancement substitutions S298T+A330M+K334V in CH2 domain andheterodimerizing substitutions K392D+K409D in the CH3 domain, while theother Fc chain has ADCC-enhancement substitutions L234Y+K290Y+Y296W inthe lower hinge region and CH2 domain along with heterodimerizingsubstitutions E356K+D399K in the CH3 domain. The antibodies prefer toform heterodimers which can induce strong ADCC killing when specificallybinding to the tumor cells with their Fab regions.

TABLE 2 Table 2: The amino acid residues located at the VH/VL and CH1/Cκinterfaces were selected for the introduction of charge pair residues.Germline residues of VH and VL are numbered by different numberingsystems, the bolded residues are the dominant ones. The contact residuesin VH/VL of most of antibodies are arrayed in the same row. Residues ofhuman IgG1 CH1 domain contacting the residues in Cκ region are alsobolded and laid in the same row. FW: Framework. VH VL IMGT # Kabat # Eu# AHo # FW Residue contact IMGT # Kabat # Eu # AHo # FW Residue 44 39 3946 2 Q/R/T

44 38 38 46 2 Q/G/H/L 49 44 44 51 2 G/R/A

* 100  100  141  4 Q/G/P * 105  109  141  4 Q/K/R/S

49 43 43 51 2 A/G/S/P C_(H)1 Cκ IMGT # Kabat # Eu # AHo # Ref # Residuecontact IMGT # Kabat # Eu # AHo # Ref # Residue 20 139 141 * 176 A

 5 116 116 * 158 F 82 175 171 * 212 P

81 162 162 * 211 S 86 188 183 * 230 S

86 176 176 * 230 S 26 145 147 * 182 K

13 124 124 * 166 Q 26 145 147 * 182 K

20 131 131 * 176 S 26 145 147 * 182 K

90 180 180 * 234 T

In a proof-of-concept study, a Fn3 tag (12 KDa) was inserted at theN-terminus of anti-EGFr Ab2 HC and a Fn3-Flag-His6 tag (14 KDa) wasfused in frame to the C-terminus of anti-EGFr Ab2 LC, so the 4 differentcombinations of LC/HC can be distinguished in SDS-PAGE gel by differentsizes: 176 KDa for the wanted LC1/HC1::LC2-Fn3-FH/Fn3-HC2 or unwantedLC2-Fn3-FH/HC1::LC1/Fn3-HC2; 162 KDa for unwanted LC1/HC1::LC1/Fn3-HC2;and 190 KDa for unwanted LC2-Fn3-FH/HC1::LC2-Fn3-FH/Fn3-HC2. Thecomposition of 176 KDa product was determined subsequently by MassSpectrometry with partial reduction. A dual-antigen binding plate ELISAwas utilized to screen the favorable variants having preferred LC/HCpairings.

Different variants (Table 3) were investigated to find the bestcombination with highest dual antigen binding and correct LC/HC pairing.When compared to the mono-specific anti-Her2 Abl (C01) or anti-EGFr Ab2(C07), which did not generate any binding signal, and the internalcontrol C11, which is made of four regular chains with random LC/HCpairings, variants 1C02, 1C04, 2A05, 2B04, 2B05 and 5D03 showed improvedbinding to dual antigens (Her2 and EGFr extracellular domain) in adose-dependent manner. Variants 2B05 and 5D03 had the strongest bindingas the curves shifted to the left most. These variants also had thedominant formation of full-length heterodimer IgG1 in the SDS-PAGE gel.Expression testing indicated that adding modifier XBP1 slightly boostedthe expression level of heterodimeric antibody variants while anothermodifier ERP23 rarely boosted expression.

TABLE 3 Variants made by electrostatic steering mechanism. The aminoacid changes in the VH/VL and CH1/CL interface of the anti-Her2 Ab1 andthe anti-EGFr Ab2 as heterodimeric IgG1 where the HC of Ab1 has S298T +A330M + K334V in CH2 domain and K392D + K409D in CH3 domain; the HC ofAb2 has L234Y + K209Y + Y296W in lower hinge and CH2 domain and E356K +D399K in CH3 domain. An Fn3 tag was inserted at the N-terminus ofanti-EGFr Ab2 HC and an Fn3-Flag-His6 tag was fused in frame to theC-terminus of anti-EGFr Ab2 LC. The residues in variable regions (VH orVL) are numbered by Kabat numbering system while residues in constantregions (CH1 or CL) are numbered by Eu numbering system. anti-Her2 Ab1HC (K392D + K409D) anti-Her2 Ab1 LC Variant VH1 CH1 VL1 CL C12 Q39K +G44K Q38D + Q100D C13 Q39K + G44K Q38D + Q100D C14 Q39K + G44K Q38D +Q100D C15 Q39K + Q105K Q38D + A43D C16 Q39K + Q105K Q38D + A43D C17Q39K + Q105K Q38D + A43D C18 G44K + Q105K Q100D + A43D C19 G44K + Q105KQ100D + A43D C20 G44K + Q105K Q100D + A43D 1A01 K147K + S183K Q124D +S176D 1A02 K147K + S183K Q124D + S176D 1A03 K147K + S183K Q124D + S176D1A04 K147K + S183K S131D + S176D 1A05 K147K + S183K S131D + S176D 1A06K147K + S183K S131D + S176D 1B01 K147K + S183K S176D + T180D 1B02K147K + S183K S176D + T180D 1B03 K147K + S183K S176D + T180D 1B04 Q39K +Q105K K147D + S183D Q38D + A43D Q124K + S176K 1B05 Q39K + Q105K K147D +S183D Q38D + A43D Q124K + S176K 1B06 Q39K + Q105K K147D + S183D Q38D +A43D Q124K + S176K 1C01 Q39K + Q105K K147D + S183D Q38D + A43D S131K +S176K 1C02 Q39K + Q105K K147D + S183D Q38D + A43D S131K + S176K 1C03Q39K + Q105K K147D + S183D Q38D + A43D S131K + S176K 1C04 Q39K + Q105KK147D + S183D Q38D + A43D S176K + T180K 1C05 Q39K + Q105K K147D + S183DQ38D + A43D S176K + T180K 1C06 Q39K + Q105K K147D + S183D Q38D + A43DS176K + T180K 1D01 K147K Q124D 1D02 K147K Q124D 1D03 K147K Q124D 1D04K147K S131D 1D05 K147K S131D 1D06 K147K S131D 2A01 S183K S176D 2A02S183K S176D 2A03 S183K S176D 2A04 Q39K + Q105K K147D Q38D + A43D Q124K2A05 Q39K + Q105K K147D Q38D + A43D Q124K 2A06 Q39K + Q105K K147D Q38D +A43D Q124K 2B01 Q39K + Q105K K147D Q38D + A43D S131K 2B02 Q39K + Q105KK147D Q38D + A43D S131K 2B03 Q39K + Q105K K147D Q38D + A43D S131K 2B04Q39K + Q105K K147D Q38D + A43D T180K 2B05 Q39K + Q105K K147D Q38D + A43DT180K 2B06 Q39K + Q105K K147D Q38D + A43D T180K 2C01 Q39K + Q105K S183DQ38D + A43D S176K 2C02 Q39K + Q105K S183D Q38D + A43D S176K 2C03 Q39K +Q105K S183D Q38D + A43D S176K 5A01 Q39K + Q105K A141D Q38D + A43D F116K5A02 Q39K + Q105K A141D Q38D + A43D F116K 5A03 Q39K + Q105K A141D Q38D +A43D F116K 5A04 Q39K + Q105K A141D Q38D + A43D F116K 5A05 Q39K + Q105KP171D Q38D + A43D S162K 5A06 Q39K + Q105K P171D Q38D + A43D S162K 5B01Q39K + Q105K P171D Q38D + A43D S162K 5B02 Q39K + Q105K P171D Q38D + A43DS162K 5B03 Q39K + Q105K K147D Q38D + A43D S131K 5B04 Q39K + Q105K K147DQ38D + A43D S131K 5B05 Q39K + Q105K K147D Q38D + A43D S131K 5B06 Q39K +Q105K K147D Q38D + A43D S131K 5C01 Q39K + Q105K S183D Q38D + A43D S176K5C02 Q39K + Q105K S183D Q38D + A43D S176K 5C03 Q39K + Q105K S183D Q38D +A43D S176K 5C04 Q39K + Q105K S183D Q38D + A43D S176K 5C05 Q39K + Q105KA141D Q38D + A43D F116K 5C06 Q39K + Q105K A141D Q38D + A43D F116K 5D01Q39K + Q105K A141D Q38D + A43D F116K 5D02 Q39K + Q105K A141D Q38D + A43DF116K 5D03 Q39K + Q105K P171D Q38D + A43D S162K 5D04 Q39K + Q105K P171DQ38D + A43D S162K 5D05 Q39K + Q105K P171D Q38D + A43D S162K 5D06 Q39K +Q105K P171D Q38D + A43D S162K 6A01 Q39K + Q105K K147D Q38D + A43D S131K6A02 Q39K + Q105K K147D Q38D + A43D S131K 6A03 Q39K + Q105K K147D Q38D +A43D S131K 6A04 Q39K + Q105K K147D Q38D + A43D S131K 6A05 Q39K + Q105KS183D Q38D + A43D S176K 6A06 Q39K + Q105K S183D Q38D + A43D S176K 6B01Q39K + Q105K S183D Q38D + A43D S176K 6B02 Q39K + Q105K S183D Q38D + A43DS176K anti-EGFr Ab2 HC (E356K + D399K) anti-EGFr Ab2 LC Variant VH2 CH1VL2 CL C12 Q39D + G44D Q38K + G100K C13 Q39D + Q105D Q38K + A43K C14G44D + Q105D G100K + A43K C15 Q39D + G44D Q38K + G100K C16 Q39D + Q105DQ38K + A43K C17 G44D + Q105D G100K + A43K C18 Q39D + G44D Q38K + G100KC19 Q39D + Q105D Q38K + A43K C20 G44D + Q105D G100K + A43K 1A01 K147D +S183D Q124K + S176K 1A02 K147D + S183D S131K + S176K 1A03 K147D + S183DS176K + T180K 1A04 K147D + S183D Q124K + S176K 1A05 K147D + S183DS131K + S176K 1A06 K147D + S183D S176K + T180K 1B01 K147D + S183DQ124K + S176K 1B02 K147D + S183D S131K + S176K 1B03 K147D + S183DS176K + T180K 1B04 G44D + Q105D S183K G100K + A43K Q124D + S176D 1B05G44D + Q105D S183K G100K + A43K S131D + S176D 1B06 G44D + Q105D S183KG100K + A43K S176D + T180D 1C01 G44D + Q105D S183K G100K + A43K Q124D +S176D 1C02 G44D + Q105D S183K G100K + A43K S131D + S176D 1C03 G44D +Q105D S183K G100K + A43K S176D + T180D 1C04 G44D + Q105D S183K G100K +A43K Q124D + S176D 1C05 G44D + Q105D S183K G100K + A43K S131D + S176D1C06 G44D + Q105D S183K G100K + A43K S176D + T180D 1D01 K147D Q124K 1D02K147D S131K 1D03 S183D S176K 1D04 K147D Q124K 1D05 K147D S131K 1D06S183D S176K 2A01 K147D Q124K 2A02 K147D S131K 2A03 S183D S176K 2A04G44D + Q105D K147K G100K + A43K Q124D 2A05 G44D + Q105D K147K G100K +A43K S131D 2A06 G44D + Q105D S183K G100K + A43K S176D 2B01 G44D + Q105DK147K G100K + A43K Q124D 2B02 G44D + Q105D K147K G100K + A43K S131D 2B03G44D + Q105D S183K G100K + A43K S176D 2B04 G44D + Q105D K147K G100K +A43K Q124D 2B05 G44D + Q105D K147K G100K + A43K S131D 2B06 G44D + Q105DS183K G100K + A43K S176D 2C01 G44D + Q105D K147K G100K + A43K Q124D 2C02G44D + Q105D K147K G100K + A43K S131D 2C03 G44D + Q105D S183K G100K +A43K S176D 5A01 Q39D + Q105D A141K Q38K + A43K F116D 5A02 Q39D + Q105DP171K Q38K + A43K S162D 5A03 Q39D + Q105D K147K Q38K + A43K S131D 5A04Q39D + Q105D S183K Q38K + A43K S176D 5A05 Q39D + Q105D A141K Q38K + A43KF116D 5A06 Q39D + Q105D P171K Q38K + A43K S162D 5B01 Q39D + Q105D K147KQ38K + A43K S131D 5B02 Q39D + Q105D S183K Q38K + A43K S176D 5B03 Q39D +Q105D A141K Q38K + A43K F116D 5B04 Q39D + Q105D P171K Q38K + A43K S162D5B05 Q39D + Q105D K147K Q38K + A43K S131D 5B06 Q39D + Q105D S183K Q38K +A43K S176D 5C01 Q39D + Q105D A141K Q38K + A43K F116D 5C02 Q39D + Q105DP171K Q38K + A43K S162D 5C03 Q39D + Q105D K147K Q38K + A43K S131D 5C04Q39D + Q105D S183K Q38K + A43K S176D 5C05 G44D + Q105D A141K G100K +A43K F116D 5C06 G44D + Q105D P171K G100K + A43K S62D 5D01 G44D + Q105DK147K G100K + A43K S131D 5D02 G44D + Q105D S183K G100K + A43K S176D 5D03G44D + Q105D A141K G100K + A43K F116D 5D04 G44D + Q105D P171K G100K +A43K S162D 5D05 G44D + Q105D K147K G100K + A43K S131D 5D06 G44D + Q105DS183K G100K + A43K S176D 6A01 G44D + Q105D A141K G100K + A43K F116D 6A02G44D + Q105D P171K G100K + A43K S162D 6A03 G44D + Q105D K147K G100K +A43K S131D 6A04 G44D + Q105D S183K G100K + A43K S176D 6A05 G44D + Q105DA141K G100K + A43K F116D 6A06 G44D + Q105D P171K G100K + A43K S162D 6B01G44D + Q105D K147K G100K + A43K S131D 6B02 G44D + Q105D S183K G100K +A43K S176D

Variants 1C02, 1C04, 2A05, 2B05 and 5D03 were made by transientlytransfecting HEK 2936E cells, and purified with a Protein A column thenpolished with Superdex 200 Size-Exclusion Column. From 900 mL ofsupernatant, 1.2-6.8 mgs of final products with ˜100% purity byanalytical SEC were obtained. In the non-reduced SDA-PAGE gel (FIG. 4,left), all variants have a very dominant full-length IgG1 in which a Fn3tag was inserted at the N-terminus of anti-EGFr Ab2 HC and aFn3-Flag-His6 tag was fused in frame to the C-terminus of anti-EGFr Ab2LC. Variants 2B05 and 5D03 are the purest with very minimal level ofsmaller bands. Under reduced condition, four different chains (Fn3-HC2at 61 KDa; HC1 at 50 KDa; LC2-Fn3-Flag-His6 at 36 KDa; LC1 at 23 KDa)were separated due to their different sizes. The four different chainswere shown to be a 1:1:1:1 ratio in the assembled full-length IgG1antibody. The components and correct LC/HC pairings were confirmed bymass spectrometry.

Example 8 Her2/EGFR Heterodimeric Antibodies Maintained BlockingFunction of the Parent Antibodies

This Example demonstrates that the heterodimeric antibodies described inExample 7 maintain the blocking function of the individual antibodiesfrom which they were made. Moreover, the heterodimeric antibodies werecapable of mediating ADCC against target-expressing cells.

In addition to dual antigen binding, variants 2B05 and 5D03 were chosento test their functionality by a cell-based assay. A CHO cell line wasstably transfected with human EGFr. When ligand EGF was added in theculture medium, the receptor EGFr on the CHO cell surface was activatedand phosphorylated, turning on downstream signal pathways, such as MAPK,ERK1/2, PI3K, JAK/STAT, and PKC. The anti-EGFr antibody from which 2B05and 5D03 were derived blocked the ligand EGF binding to the receptorEGFr and inhibited the phosphorylation of receptor EGFr at IC50=2.7 nM.The combo of anti-EGFr antibody and anti-Her2 antibody functionedsimilarly at IC50=3.2 nM. Anti-EGFr×Her2 heterodimeric antibody variants2B05 and 5D03 both were comparable in the phosphorylation of receptorEGFr at IC50=4.2 nM and IC50=4.6 nM respectively, indicating theanti-EGFr Fab arm of heterodimeric antibody variants 2B05 and 5D03 wasfunctioning comparable to the wild type anti-EGFr antibody.

BT474 is a human breast tumor cell line. BT474 cells express both Her2(Erb2) and Her3 (Erb3) on the surface. Anti-Her2 antibodies can bind todomain IV of Her2, triggering internalization and degradation ofreceptors [Wehrman T S, et al. (2006) PNAS 103(50):19063-19068 andBuschenfelde C M et al (2002) Cancer Res 62(8):2244-2247]. The antibodythus blocks downstream signaling pathways [Yakes F M, et al. (2002)Cancer Res 62(14):4132-4141 and Scotti M L et al (2008) Breast CancerRes Treat 111(2):241-50], such as Raf/MEK 1&2/ERK 1&2 and PI3K/Aktpathways. The anti-Her2 antibody does not decrease Her2 phosphorylationbut inhibits basal Her3 phosphorylation in BT474 cells. When no ligandis added in the culture medium of BT474 cells, the Her2 antibody aloneblocked the phosphorylation of Her3 at IC50=2.8 nM. The combination ofanti-EGFr antibody and anti-Her2 antibody functioned similarly atIC50=5.2 nM. Anti-EGFr×Her2 heterodimeric antibody variants 2B05 and5D03 both inhibited the basal level phosphorylation of receptor Her3 atIC50=3.0 nM and IC50=3.6 nM respectively, indicating that the anti-Her2arm was functioning.

The combined data from the above two different cell-based assayssuggested that both arms of anti-EGFr×Her2 heterodimeric antibodyvariants 2B05 and 5D03 work properly in inhibiting the activation ofEGFr and Her2.

Example 9 Her2/EGFR Heterodimeric Antibodies are Capable of MediatingADCC Killing of Tumor Cells

N87 is a human gastric tumor cell line expressing high levels of Her2and moderate levels of EGFr. An irrelevant human IgG1 was used as acontrol. The anti-EGFr×Her2 heterodimeric antibody variants 2B05 and5D03 have incorporated ADCC enhancement substitutions andheterodimerizing substitutions in their Fc regions. The ADCC assay wascarried out to test whether the heterodimeric antibody variants bind totheir specific antigens and induce killing of N87 cells. At 1 μg/mL theirrelevant human IgG1 control antibody had a background lysis of 30% anddid not show a dose-dependent response when it was titrated down. Bothvariants 2B05 and 5D03 had much higher specific lysis at 1 μg/mLconcentration and showed a dose-dependent response with EC50 at 0.10 pMand 0.19 pM respectively. The data demonstrates that the heterodimericantibody variants 2B05 and 5D03 can bind to targets EGFr & Her2, andinduce strong killing of N87 cells by engaging NK cells.

Example 10 Alternative Variants can Also Guide Correct LC/HC Pairings

Electrostatic steering can be combined with other steering technologies.For example, replacement of one charged-residue pair in VH/VL interfacewith a pair of cysteine residues was explored. The pair of cysteineresidues are in close proximity (4˜5.6 Å) to form disulfide bond,therefore locking the correctly paired LC/HC. Seven differentcombinations of charge-pair and Cys-Cys pair on the basis of variant2B05 (Table 4) were made by transiently transfecting mammalian 2936Ecells. Supernatants were separated by an SDS-PAGE gel. While theinternal control C11 showed four different bands between 148 and 250KDa, variants 2C04 and 2C06 dominantly produced the full-length IgG1;variant 2D04 which has different disulfide bonds at Q105C-A43C andG44C-G100C in separate Fab arms exclusively assembles into thefull-length IgG1 antibody, implying that the combination of charge pairresidues and cysteine pair residues can work cooperatively to make thecorrectly paired and folded heterodimeric antibody.

TABLE 4 Variants made by the combination of charge pair residues andCysteine pair residues in the variable regions. The residues in variableregions (VH or VL) are numbered by Kabat numbering system while residuesin constant regions (CH1 or CL) are numbered by Eu numbering system. AFn3 tag is inserted at the N-terminus of anti-EGFr Ab2 HC and aFn3-Flag-His6 tag is fused in frame to the C-terminus of anti-EGFr Ab2LC. anti-Her2 Ab1 HC anti-Her2 anti-EGFr Ab2 HC anti-EGFr (K392D +K409D) Ab1 LC (E356K + D399K) Ab2 LC Variant VH1 C_(H)1 VL1 CL VH2C_(H)1 VL2 CL 2C04 Q39C + Q105K K147D Q38C + A43D T180K G44D + Q105DK147K G100K + A43K S131D 2C05 Q39K + Q105C K147D Q38D + A43C T180KG44D + Q105D K147K G100K + A43K S131D 2C06 Q39K + Q105K K147D Q38D +A43D T180K G44C + Q105D K147K G100C + A43K S131D 2D01 Q39K + Q105K K147DQ38D + A43D T180K G44D + Q105C K147K G100K + A43C S131D 2D02 Q39C +Q105K K147D Q38C + A43D T180K G44C + Q105D K147K G100C + A43K S131D 2D03Q39C + Q105K K147D Q38C + A43D T180K G44D + Q105C K147K G100K + A43CS131D 2D04 Q39K + Q105C K147D Q38D + A43C T180K G44C + Q105D K147KG100C + A43K S131D

Replacement of a pair of charged residues in VH/VL interface with a pairof bulky/small residues [Zamyatnin A A (1972) Prog. Biophos. Mol. Biol.24:107-123; Chothia C (1975) J. Mol. Biol. 105:1-14] was also tested.The bulky/small residue pairs could exert a knob-into-hole effect,directing the correct LC/HC pairings in the combination of electrostaticsteering mechanism. Bulky residues, for example, Tryptophan (W) has avolume of 227.8 Å3 and an Accessible Surface Area of 255 Å2; Tyrosine(Y) has a volume of 193.6 A3 and an Accessible Surface Area of 230 Å2.Small residue Alanine (A) has only a volume of 88.6 Å3 and an AccessibleSurface Area of 115 Å2 while Serine (S) is similarly having a volume of89 Å3 and an Accessible Surface Area of 115 Å2. A series of 64 variants(Table 5) on the basis of heterodimeric antibody variant 2B05 were madeand tested by Western blotting. Variants 3A01, 3A02, 3A03, 3A04, 3A05,3A06, 3B01, 3B02, 3B03, 3B04, 8A03 and 8A04 exclusively show a singleband after separation by an SDS-PAGE gel, indicating that the fourdifferent chains can assemble into a full-length heterodimeric antibody.Other variants had multiple bands after separation by an SDS-PAGE gel,suggesting that these LCs could have some issues in terms of pairingwith their cognate HCs.

TABLE 5 Variants made by the combination of charge pair residues andbulky/small pair residues in the variable regions. The bulky residueshere represent Tryptophan (W) or Tyrosine (Y) while the small residuesrepresent Alanine (A) or Serine (S). The residues in variable regions(VH or VL) are numbered by Kabat numbering system while residues inconstant regions (CH1 or CL) are numbered by Eu numbering system. A Fn3tag is inserted at the N-terminus of anti-EGFr Ab2 HC and aFn3-Flag-His6 tag is fused in frame to the C-terminus of anti-EGFr Ab2LC. anti-Her2 Ab1 HC anti-Her2 anti-EGFr Ab2 HC anti-EGFr (K392D +K409D) Ab1 LC (E356K + D399K) Ab2 LC Variant VH1 CH1 VL1 CL VH2 CH1 VL2CL 3A01 Q39A + Q105K K147D Q38W + A43D T180K G44D + Q105W K147K G100K +A43S S131D 3A02 Q39A + Q105K K147D Q38Y + A43D T180K G44D + Q105W K147KG100K + A43S S131D 3A03 Q39S + Q105K K147D Q38W + A43D T180K G44D +Q105W K147K G100K + A43S S131D 3A04 Q39S + Q105K K147D Q38Y + A43D T180KG44D + Q105W K147K G100K + A43S S131D 3A05 Q39K + Q105A K147D Q38D +A43W T180K G44D + Q105W K147K G100K + A43S S131D 3A06 Q39K + Q105A K147DQ38D + A43Y T180K G44D + Q105W K147K G100K + A43S S131D 3B01 Q39K +Q105S K147D Q38D + A43W T180K G44D + Q105W K147K G100K + A43S S131D 3B02Q39K + Q105S K147D Q38D + A43Y T180K G44D + Q105W K147K G100K + A43SS131D 3B03 Q39W + Q105K K147D Q38A + A43D T180K G44D + Q105W K147KG100K + A43S S131D 3B04 Q39W + Q105K K147D Q38S + A43D T180K G44D +Q105W K147K G100K + A43S S131D 3B05 Q39Y + Q105K K147D Q38A + A43D T180KG44D + Q105W K147K G100K + A43S S131D 3B06 Q39Y + Q105K K147D Q38S +A43D T180K G44D + Q105W K147K G100K + A43S S131D 3C01 Q39K + Q105W K147DQ38D + A43S T180K G44D + Q105W K147K G100K + A43S S131D 3C02 Q39K +Q105Y K147D Q38D + A43S T180K G44D + Q105W K147K G100K + A43S S131D 3C03Q39A + Q105K K147D Q38W + A43D T180K G44D + Q105Y K147K G100K + A43SS131D 3C04 Q39A + Q105K K147D Q38Y + A43D T180K G44D + Q105Y K147KG100K + A43S S131D 3C05 Q39S + Q105K K147D Q38W + A43D T180K G44D +Q105Y K147K G100K + A43S S131D 3C06 Q39S + Q105K K147D Q38Y + A43D T180KG44D + Q105Y K147K G100K + A43S S131D 3D01 Q39K + Q105A K147D Q38D +A43W T180K G44D + Q105Y K147K G100K + A43S S131D 3D02 Q39K + Q105A K147DQ38D + A43Y T180K G44D + Q105Y K147K G100K + A43S S131D 3D03 Q39K +Q105S K147D Q38D + A43W T180K G44D + Q105Y K147K G100K + A43S S131D 3D04Q39K + Q105S K147D Q38D + A43Y T180K G44D + Q105Y K147K G100K + A43SS131D 3D05 Q39W + Q105K K147D Q38A + A43D T180K G44D + Q105Y K147KG100K + A43S S131D 3D06 Q39W + Q105K K147D Q38S + A43D T180K G44D +Q105Y K147K G100K + A43S S131D 4A01 Q39Y + Q105K K147D Q38A + A43D T180KG44D + Q105Y K147K G100K + A43S S131D 4A02 Q39Y + Q105K K147D Q38S +A43D T180K G44D + Q105Y K147K G100K + A43S S131D 4A03 Q39K + Q105W K147DQ38D + A43S T180K G44D + Q105Y K147K G100K + A43S S131D 4A04 Q39K +Q105Y K147D Q38D + A43S T180K G44D + Q105Y K147K G100K + A43S S131D 7A01Q39A + Q105K K147D Q38W + A43D T180K G44D + Q105D K147K G100K + A43KS131D 7A02 Q39A + Q105K K147D Q38Y + A43D T180K G44D + Q105D K147KG100K + A43K S131D 7A03 Q39S + Q105K K147D Q38W + A43D T180K G44D +Q105D K147K G100K + A43K S131D 7A04 Q39S + Q105K K147D Q38Y + A43D T180KG44D + Q105D K147K G100K + A43K S131D 7A05 Q39K + Q105A K147D Q38D +A43W T180K G44D + Q105D K147K G100K + A43K S131D 7A06 Q39K + Q105A K147DQ38D + A43Y T180K G44D + Q105D K147K G100K + A43K S131D 7B01 Q39K +Q105S K147D Q38D + A43W T180K G44D + Q105D K147K G100K + A43K S131D 7B02Q39K + Q105S K147D Q38D + A43Y T180K G44D + Q105D K147K G100K + A43KS131D 7B03 Q39W + Q105K K147D Q38A + A43D T180K G44D + Q105D K147KG100K + A43K S131D 7B04 Q39W + Q105K K147D Q38S + A43D T180K G44D +Q105D K147K G100K + A43K S131D 7B05 Q39Y + Q105K K147D Q38A + A43D T180KG44D + Q105D K147K G100K + A43K S131D 7B06 Q39Y + Q105K K147D Q38S +A43D T180K G44D + Q105D K147K G100K + A43K S131D 7C01 Q39K + Q105W K147DQ38D + A43S T180K G44D + Q105D K147K G100K + A43K S131D 7C02 Q39K +Q105Y K147D Q38D + A43S T180K G44D + Q105D K147K G100K + A43K S131D 7C03Q39K + Q105K K147D Q38D + A43D T180K G44A + Q105D K147K G100W + A43KS131D 7C04 Q39K + Q105K K147D Q38D + A43D T180K G44A + Q105D K147KG100Y + A43K S131D 7C05 Q39K + Q105K K147D Q38D + A43D T180K G44S +Q105D K147K G100W + A43K S131D 7C06 Q39K + Q105K K147D Q38D + A43D T180KG44S + Q105D K147K G100Y + A43K S131D 7D01 Q39K + Q105K K147D Q38D +A43D T180K G44D + Q105A K147K G100K + A43W S131D 7D02 Q39K + Q105K K147DQ38D + A43D T180K G44D + Q105A K147K G100K + A43Y S131D 7D03 Q39K +Q105K K147D Q38D + A43D T180K G44D + Q105S K147K G100K + A43W S131D 7D04Q39K + Q105K K147D Q38D + A43D T180K G44D + Q105S K147K G100K + A43YS131D 7D05 Q39K + Q105K K147D Q38D + A43D T180K G44W + Q105D K147KG100A + A43K S131D 7D06 Q39K + Q105K K147D Q38D + A43D T180K G44W +Q105D K147K G100S + A43K S131D 8A01 Q39K + Q105K K147D Q38D + A43D T180KG44Y + Q105D K147K G100A + A43K S131D 8A02 Q39K + Q105K K147D Q38D +A43D T180K G44Y + Q105D K147K G100S + A43K S131D 8A03 Q39K + Q105K K147DQ38D + A43D T180K G44D + Q105W K147K G100K + A43S S131D 8A04 Q39K +Q105K K147D Q38D + A43D T180K G44D + Q105Y K147K G100K + A43S S131D 8A05Q39A + Q105K K147D Q38W + A43D T180K G44W + Q105D K147K G100A + A43KS131D 8A06 Q39A + Q105K K147D Q38W + A43D T180K G44Y + Q105D K147KG100A + A43K S131D 8B01 Q39A + Q105K K147D Q38W + A43D T180K G44W +Q105D K147K G100S + A43K S131D 8B02 Q39A + Q105K K147D Q38W + A43D T180KG44Y + Q105D K147K G100S + A43K S131D 8B03 Q39W + Q105K K147D Q38A +A43D T180K G44A + Q105D K147K G100W + A43K S131D 8B04 Q39W + Q105K K147DQ38A + A43D T180K G44A + Q105D K147K G100Y + A43K S131D 8B05 Q39Y +Q105K K147D Q38A + A43D T180K G44A + Q105D K147K G100W + A43K S131D 8B06Q39Y + Q105K K147D Q38A + A43D T180K G44A + Q105D K147K G100Y + A43KS131D

Example 11 Optimization of Heterodimeric Antibodies in the Absence ofTags

The tags of anti-EGFr×Her2 variants 2B05 and 5D03 were removed andtested by either transfecting mammalian 2936E cells with four DNAs tomake full-length antibody, or with only two DNAs to assess the toleranceof mismatched LC/HC pairings. When all four different chains werepresent, both variants produced the full-length antibody with asignificant amount of half-antibody. Transfections with two plasmidsencoding matched LC1+HC1 or LC2+HC2 also produced the full-lengthhomodimer antibody with a significant amount of half-antibody. When theLC2 were mispaired with their non-cognate HCl (LC2+HC1), there was smallamount of products shown in the gel, whereas no products formed when LC1was mis-paired with its non-cognate HC2 (LC1+HC2). The expressiontesting implied that LC2 of the anti-EGFr antibody is tolerated with HClof the anti-Her2 antibody, while LC1 of the anti-Her2 antibody is nottolerated with HC2 of the anti-EGFr antibody. To maximize theelectrostatic steering effect, a series of new variants (Table 6) wereinvestigated by introducing charge-pair residues at the same spatialposition, but with opposite polarity. The LC/HC interfaces are mutuallyreciprocal, either repulsive when the same polarity residues come close,or attractive when the opposite polarity residues are in proximity.

Variants V15 and V20 were mainly expressed as the intact antibody afterfour different chains were transcribed and translated. In the presenceof matched HC/LC (i.e. LC1+HC1 or LC2+HC2), half-antibody and homodimerfull-length antibody were produced. When mismatched HC/LC (e.g. LC2+HC1)were co-expressed, no product was observed, suggesting that the LC2 wasnot compatible with HCl. However, LC1 was tolerated by HC2 to formeither half-antibody or homodimer antibody, implying that the LC1 of V15and V20 can pair with the non-cognate HC2 and get assembled thensecreted. In contrast, variants V21, V23 and V25 were mainly expressedas the intact antibody after four different chains were transcribed andtranslated. In the presence of matched two chains (i.e. LC1+HC1 orLC2+HC2), a full IgG antibody was produced, indicating the LCs arecompatible with their cognate HCs. In the presence of mismatched LC2+HClor LC1+HC2, no product was formed, suggesting that the LCs were nottolerated with the non-cognate HCs. Variant V22 was not as effective asV21, V23 and V25, as minor products were observed when the mismatchedLCs were forced to pair with the non-cognate HCs. Mass spectrometryanalysis demonstrated that variants V12, V21, V23, and V25 have thepresence of four different chains (LC1+HC1+LC2+HC2) and correct LC/HCpairings (LC1+HCl and LC2+HC2).

TABLE 6 The amino acid changes in the VH/VL and CH1/CL interface ofanti- Her2 Ab1 and anti-EGFr Ab2 as heterodimeric IgG1 with mutualrepulsive/attractive mechanism. The HC of anti-Her2 Ab1 has S298T +A330M + K334V in CH2 domain and K392D + K409D in CH3 domain; the HC ofanti-EGFr Ab2 has L234Y + K209Y + Y296W in lower hinge/CH2 domain andE356K + D399K in CH3 domain. The residues in variable regions (VH or VL)are numbered by Kabat numbering system while residues in constantregions (CH1 or CL) are numbered by Eu numbering system. anti-Her2 Ab1HC anti-Her2 anti-EGFr Ab2 HC anti-EGFr (K392D + K409D) Ab1 LC (E356K +D399K) Ab2 LC Variant VH1 CH1 VL1 CL VH2 CH1 VL2 CL V01 Q39K + Q105KK147D Q38D + A43D T180K G44D + Q105D K147K G100K + A43K S131D V02*Q39K + Q105C K147D Q38D + A43C T180K G44C + Q105D K147K G100C + A43KS131D V03 Q39K + Q105K P171D Q38D + A43D S162K G44D + Q105D A141KG100K + A43K F116D V04 Q39K + Q105K P171D Q38D + A43D S162K Q105D A141KA43K F116D V05 Q39K + Q105K P171D Q38D + A43D S162K Q39D + Q105D A141KQ38K + A43K F116D V06 Q39K + Q105K K147D Q38D + A43D T180K G44D + Q105DG100K + A43K V07 Q39K P171D Q38D S162K Q39D A141K Q38K F116D V08 Q39KA141D Q38D F116K Q39D P171K Q38K S162D V09 Q39K A141D Q38D F116K Q39DA141K Q38K F116D V10 Q39K P171D Q38D S162K Q39D P171K Q38K S162D V11Q105K A141D A43D F116K Q105D A141K A43K F116D V12 Q105K P171D A43D S162KQ105D P171K A43K S162D V13 Q105K A141D A43D F116K Q105D P171K A43K S162DV14 Q105K P171D A43D S162K Q105D A141K A43K F116D V15 Q39K S183D Q38DS176K Q39D S183K Q38K S176D V16 Q105K S183D A43D S176K Q105D S183K A43KS176D V17 Q39K S183D Q38D S176K Q105D S183K A43K S176D V18 Q105K S183DA43D S176K Q39D S183K Q38K S176D V19 Q39K A141D + Q38D F116K + Q39DA141K + Q38K F116D + S183K S176D S183D S176K V20 Q39K A141D + Q38DF116K + Q39D A141K + Q38K F116D + P171K S162D P171D S162K V21 Q39K +Q105K K147D Q38D + A43D T180K Q39D + Q105D K147K Q38K + A43K T180D V22Q39K + Q105K K147D Q38D + A43D T180K Q39D + Q105D K147K Q38K + A43KS131D V23 Q39K + Q105K S183D Q38D + A43D S176K Q39D + Q105D S183K Q38K +A43K S176D V24 Q39K + Q105K P171D Q38D + A43D S162K Q39D + Q105D P171KQ38K + A43K S162D V25 G44K + Q105K S183D Q100D + A43D S176K G44D + Q105DS183K G100K + A43K S176D

Example 12 Optimized EGFR/Her2 Heterodimeric Antibody Variants ShowedThermal Stability

The temperature-induced unfolding of anti-EGFr IgG2, anti-Her2 IgG1,anti-Her2 defucosylated IgG1 and four anti-EGFr×Her2 heterodimericantibody variants, under the same solvent conditions were assessed bydifferential scanning calorimetry (DSC). The thermogram of each proteinconsisted of 2 or 3 transitions. Wild-type anti-Her2 antibody showed aTm of Fab/CH3 at 82° C. and a Tm of CH2 at 72° C.; an afucosylatedversion of the anti-Her2 antibody did not change the Tm of separatedomains but decreased the enthalpy slightly. The anti-EGFr antibody hada similar profile of temperature-induced unfolding. All fouranti-EGFr×Her2 heterodimeric antibody variants had slightly decreased Tmof CH2/CH3 at 68° C. as they all have the ADCC-enhancement substitutionsin CH2 domains and heterodimerizing substitutions in the CH3 domain. Interms of Tm of Fab domains, variant V12 and V24 had the most significantdecrease from 82° C. to 72° C.; variant V25 had two separate peaks at72° C. and 78° C. while variant V23 had a single peak at 78° C. Overall,the four heterodimeric antibody variants showed good thermal stability.The data suggested the selected positions for introducing charge pairresidues in the Fab regions impact on the stability of intactheterodimeric antibody to some extent, with Tm of separate domains above68° C.

Example 13 Heterodimeric Antibodies Targeting Two Different Epitopes ofthe Same Antigen

In order to show that the same approach of making heterodimericantibodies can be applied to different antibodies, a heterodimeric IgGwas generated from two different anti-Her2 antibodies. One antibodybinds to the domain IV of Her2 whereas the other binds to domain II ofHer2. Variant V23 (Table 6) in which two pairs of charge residues inVH/VL and one pair of charge residues in CH1/CL were reciprocallyintroduced was tested by either transfecting with four DNAs to makefull-length antibody, or with only two DNAs to assess the tolerance ofmismatched LC/HC pairings. Similarly to anti-EGFr×Her heterodimericantibody variant V23, the anti-Her2×Her2 heterodimeric antibody V23 wasmainly expressed as the intact antibody after four different chains weretranslated and assembled. In the presence of two matched chains (i.e.LC1+HC1 or LC2+HC2), half-antibody and homodimer antibody were produced,indicating that the LCs are compatible with their cognate HCs. In thepresence of mismatched chains LC2+HC1 or LC1+HC2, absolutely no productwas formed, suggesting that the LCs were not tolerated with theirnon-cognate HCs.

Example 14 Different Combinations of Charged Residues AffectHeterodimeric Antibody Expression and LC/HC Pairings

To investigate whether different combinations of charge residues leadsto different expression level or affects the LC/HC pairings, severalanti-Her2×Her2 heterodimeric antibody variants were made by introducingcharge pair residues with different combinations at the same locations(Table 7). While V23B had correct LC/HC pairings like V23A, but theexpression level went down either in the form of intact antibody or halfantibody. However, V23C and V23D tolerated the mispairing of LC/HC. Alltogether, this set of data suggested that the electrostatic steering isnot the only factor to guide the correct LC/HC pairings; othermechanisms such as shape complimentarity may play a role in the process.

TABLE 7 Comparison of different charge pair combinations at the sameposition of VH/VL and CH1/CL interfaces. The residues in variableregions (VH or VL) are numbered by Kabat numbering system while residuesin constant regions (CH1 or CL) are numbered by Eu numbering system. TheHC of anti-Her2 Ab1 has ADCC-enhancement substitutions S298T + A330M +K334V in CH2 domain and heterodimerizing changes K392D + K409D in CH3domain; the HC of anti-Her2 Ab2 has ADCC-enhancement substitutionsL234Y + K209Y + Y296W in lower hinge/CH2 domain and heterodimerizingchanges E356K + D399K in CH3 domain. anti-Her2 Ab1 HC anti-Her2anti-Her2 Ab2 HC anti-Her2 (K392D + K409D) Ab1 LC (E356K + D399K) Ab2 LCVariant VH1 CH1 VL1 CL VH2 CH1 VL2 CL V23A Q39K + Q105K S183D Q38D +A43D S176K Q39D + Q105D S183K Q38K + A43K S176D V23B Q39D + Q105K S183KQ38K + A43D S176D Q39K + Q105D S183D Q38D + A43K S176K V23C Q39K + Q105DS183K Q38D + A43K S176D Q39D + Q105K S183D Q38K + A43D S176K V23D Q39D +Q105D S183K Q38K + A43K S176D Q39K + Q105K S183D Q38D + A43D S176K

Example 15 Stability and Viscosity Studies

Stability studies were conducted for a compositions comprisingrepresentative heterodimeric antibodies described herein, and featuresof the antibody composition were compared to comparable DVD (dualvariable domain) antibody compositions. Antibody samples were stored at4° C. or 40° C. in an A52Su formulation (i.e., 10 mM Acetate, 9%Sucrose, pH 5.2) for a period of two weeks or two months. Antibodyaggregation was measured as a surrogate for stability using, e.g.,SE-HPLC, CEX-HPLC, HIAC (sub-visible particle), and visual inspection.The results are summarized as percent of the monomeric peak (i.e., 100%would indicate no observed aggregation) in Table 8.

TABLE 8 % monomeric Antibody % monomeric peak at peak at 40° C. typeAntibody Name 4° C. for two weeks for two weeks hetero-Ig Ab-5-6.147v199.2 95.3 hetero-Ig Ab-23-6.147v1 99.6 98.0 hetero-Ig Ab-23-6.147v2 99.798.7 hetero-Ig Ab-5-6.37.5v1 99.6 96.1 hetero-Ig Ab-23-6.37.5v1 99.698.7 DVD Ab-23-6.147 N/A 62 DVD Ab-5-6.147 N/A 15 DVD Ab-23-6.37.5 N/A89 DVD Ab-20C3-6.147 N/A 53

As demonstrated by the results set forth in Table 8, less than 5% of thehetero-Ig antibodies in A52Su formed aggregates when stored at 40° C.for two weeks compared to the DVD antibodies tested, where 11%-85% ofthe DVD antibodies in A52Su formed aggregates when stored under similarconditions. When stored at 4° C. for two weeks, less than 1% of thehetero-Ig antibodies tested formed aggregates.

A separate study was performed to investigate the relationship betweenviscosity and varying concentrations (70 mg/mL or 150 mg/mL) of the DVDand heterodimeric antibodies in the A52Su formulation. Viscosity wasmeasured using a rheometer with the cone/plate geometry (RV III+ model,Brookfield Engineering Labs, Inc., Middleboro, Mass.). Sampletemperature was maintained at 25° C. during measurement with a waterbath. The spindle speed ranged from 15 to 125 rpm with 10 rpmincrements. Data collection was carried out with Rheocalc™ software,version 2.7. The viscosity measurements of the various antibodies testedin A52Su formulations are provided below in Table 9.

TABLE 9 Viscosity (cP) Viscosity (cP) Antibody type Antibody Name at 70mg/mL at 150 mg/mL hetero-Ig Ab-5-6.147v1 4.5 35.7 hetero-IgAb-23-6.147v1 3.5 15.6 hetero-Ig Ab-23-6.147v2 4.2 16.8 hetero-IgAb-5-6.37.5v1 3.1 51.5 hetero-Ig Ab-23-6.37.5v1 2.1 13.7 DVD Ab-23-6.1474.3 94 DVD Ab-5-6.147 4.4 65 DVD Ab-23-6.37.5 4.6 43 DVD Ab-20C3-6.1474.6 15

As shown in Table 9, the viscosity of each of the compositionscomprising one of the tested heterodimeric or DVD antibodies (70 mg/mL)was less than 6 cP. When the heterodimeric antibody was present in thecomposition at a concentration of 150 mg/mL, three of the compositionshad a viscosity of less than 17 cP. In contrast to the heterodimericantibody formulations, only one DVD antibody formulation had a viscosityof less than 16 cP when the DVD antibody was present at a concentrationof 150 mg/mL.

The foregoing Example demonstrates that the heterodimeric antibodiesdescribed herein are more stable (i.e., less than 1% of the antibody inthe composition form aggregates when stored under 4° C. for two weeks)than the bispecific DVD antibodies tested. Formulations comprising suchheterodimeric antibodies also met preferred viscosity specifications,and thus are particularly suitable for large scale manufacturing.

1. A heterodimeric antibody or fragment thereof comprising one or moresubstitutions in each of the following domains: a first CH3-domain, asecond CH3-domain, a CH1-domain, a CL-domain, a VH-domain and aVL-domain, wherein the one or more substitutions introduce charged aminoacids that are electrostatically unfavorable to homodimer formation andelectrostatically favorable to heterodimer formation.
 2. Theheterodimeric antibody or fragment thereof of claim 1, wherein the firstCH3-domain or the second CH3-domain comprises an amino acid sequencediffering from wild-type IgG amino acid sequence such that (a) one ormore positive-charged amino acids in a wild-type human IgG amino acidsequence are replaced with one or more negative-charged amino acids or(b) one or more negative-charged amino acids in a wild-type human IgGamino acid sequence are replaced with one or more positive-charged aminoacids.
 3. (canceled)
 4. The heterodimeric antibody or fragment thereofof claim 1, wherein the CH1-domain or the CL-domain comprises an aminoacid sequence differing from wild-type IgG amino acid sequence such that(a) one or more positive-charged amino acids in wild-type IgG amino acidsequence are replaced with one or more negative-charged amino acids or(b) one or more negative-charged amino acids in wild-type IgG amino acidsequence are replaced with one or more positive-charged amino acids. 5.(canceled)
 6. The heterodimeric antibody or fragment thereof of claim 1,wherein the VH-domain or the VL-domain comprises an amino acid sequencediffering from wild-type IgG amino acid sequence such that (a) one ormore positive-charged amino acids in wild-type IgG amino acid sequenceare replaced with one or more negative-charged amino acids or (b) one ormore negative-charged amino acids in wild-type IgG amino acid sequenceare replaced with one or more positive-charged amino acids. 7.(canceled)
 8. The heterodimeric antibody or fragment thereof of claim 1,wherein the positive-charged amino acid is selected from the groupconsisting of lysine, histidine and arginine.
 9. The heterodimericantibody or fragment thereof of claim 1, wherein the negative-chargedamino acid is selected from the group consisting of aspartic acid andglutamic acid.
 10. The heterodimeric antibody or fragment thereof ofclaim 1, wherein the first CH3-domain comprises a replacement of aglutamic acid with a positive-charged amino acid.
 11. (canceled)
 12. Theheterodimeric antibody or fragment thereof of claim 1, wherein the firstCH3-domain comprises a replacement of an aspartic acid with anegative-charged amino acid.
 13. (canceled)
 14. The heterodimericantibody or fragment thereof of claim 1, wherein the second CH3-domaincomprises a replacement of a lysine with a negative-charged amino acid.15. (canceled)
 16. The heterodimeric antibody or fragment thereof ofclaim 1, wherein the CH1-domain comprises a replacement of a serine witha negative-charged amino acid.
 17. (canceled)
 18. The heterodimericantibody or fragment thereof of claim 1, wherein the CL-domain comprisesa replacement of a serine with a positive-charged amino acid. 19.(canceled)
 20. The heterodimeric antibody or fragment thereof of claim1, wherein the VH-domain comprises a replacement of a glutamine with anegative-charged amino acid.
 21. (canceled)
 22. The heterodimericantibody or fragment thereof of claim 1, wherein the VL-domain comprisesa replacement of a glutamine with a positive-charged amino acid. 23.(canceled)
 24. The heterodimeric antibody or fragment thereof of claim1, that is selected from the group consisting of an antibody, abispecific antibody, a monospecific monovalent antibody, a bispecificmaxibody, a monobody, a peptibody, a bispecific peptibody, a monovalentpeptibody and a receptor fusion protein.
 25. A heterodimeric antibody orfragment thereof comprising a heavy chain comprising (a) a first aminoacid substitution at an AHo position selected from the group consistingof AHo positions 42-50 that introduces a charged amino acid at saidposition, (b) a second amino acid substitution at a position selectedfrom the group consisting of positions 126-213 (EU numbering) thatintroduces a charged amino acid at said position, (c) a third amino acidsubstitution at a position selected from the group consisting ofpositions 352-360 (EU numbering) that introduces a charged amino acid atsaid position, and (d) a fourth amino acid substitution at a positionselected from the group consisting of positions 395-403 (EU numbering)that introduces a charged amino acid, wherein the charged amino acid of(a) has the same charge as the charged amino acid of (b), and whereinthe charged amino acids of (c) and (d) have an opposite charge of thecharged amino acids of (a) and (b).
 26. The heterodimeric antibody orfragment thereof of claim 25, wherein the first amino acid substitutionis at AHo position 46, the second amino acid substitution is at EUposition 183, the third amino acid substitution is at EU position 356and the fourth amino acid substitution is at EU position
 399. 27. Theheterodimeric antibody or fragment thereof of claim 25, whereinglutamine at AHo position 46 is replaced with glutamic acid, serine atEU position 183 is replaced with glutamic acid, glutamic acid at EUposition 356 is replaced with lysine, and aspartic acid at EU position399 is replaced with lysine.
 28. An antibody or fragment thereofcomprising a heavy chain comprising (a) a first amino acid substitutionat an AHo position selected from the group consisting of AHo positions35-43 that introduces a charged amino acid at said position, (b) asecond amino acid substitution at a position selected from the groupconsisting of positions 126-213 (EU numbering) that introduces a chargedamino acid at said position, (c) a third amino acid substitution at aposition selected from the group consisting of positions 388-398 (EUnumbering) that introduces a charged amino acid at said position, and(d) a fourth amino acid substitution at a position selected from thegroup consisting of positions 404-413 (EU numbering) that introduces acharged amino acid, wherein the charged amino acids of (c) and (d) havean opposite charge of the charged amino acids of (a) and (b).
 29. Theheterodimeric antibody or fragment thereof according to claim 28,wherein the first amino acid substitution is at AHo position 46, thesecond amino acid substitution is at EU position 183, the third aminosubstitution is at EU position 393 and the fourth amino acidsubstitution is at EU position
 409. 30. The antibody or fragment thereofclaim 29, wherein glutamine at AHo position 46 is replaced with lysine,serine at EU position 183 is replaced with lysine, lysine at EU position392 is replaced with aspartic acid and lysine at EU position 409 isreplaced with aspartic acid.
 31. An heterodimeric antibody or fragmentcomprising a light chain comprising (a) a first amino acid substitutionat an AHo position selected from the group consisting of AHo positions42-50 that introduces a charged amino acid at said position, and (b) asecond amino acid substitution at a position selected from the groupconsisting of positions 126-213 (EU numbering) that introduces a chargedamino acid at said position.
 32. The heterodimeric antibody or fragmentthereof of claim 31, wherein the first amino acid substitution is at AHoposition 46 and the second amino acid substitution is at EU position176.
 33. The heterodimeric antibody or fragment of claim 32, whereinglutamine at AHo position 46 is replaced with glutamic acid and serineat EU position 176 is replaced with glutamic acid.
 34. The heterodimericantibody or fragment thereof of claim 25, wherein (a) and (b) have anegative-charge and the charged amino acids of (c) and (d) have apositive charge.
 35. (canceled)
 36. The antibody or fragment thereof ofclaim 34, comprising a light chain comprising positive-charged aminoacids at AHo position 46 and EU position
 176. 37. A heterodimericantibody or fragment thereof comprising a heavy chain comprising (a) afirst amino acid substitution at an AHo position selected from the groupconsisting of AHo positions 42-50 that introduces a charged amino acidat said position, (b) a second amino acid substitution at a positionselected from the group consisting of positions 126-213 (EU numbering)that introduces a charged amino acid at said position, (c) a third aminoacid substitution at a position selected from the group consisting ofpositions 388-397 (EU numbering) that introduces a charged amino acid atsaid position, and (d) a fourth amino acid substitution at a positionselected from the group consisting of positions 404-413 (EU numbering)that introduces a charged amino acid, wherein the charged amino acids of(a) and (b) have a positive-charge and the charged amino acids of (c)and (d) have a negative-charge.
 38. The heterodimeric antibody orfragment thereof of claim 37, wherein the first amino acid substitutionis at AHo position 46, the second amino acid substitution is at EUposition 183, the third amino acid substitution is at EU position 392and the fourth amino acid substitution is at EU position
 409. 39. Theheterodimeric antibody or fragment thereof of claim 37, comprising alight chain comprising negative-charged amino acids at AHo position 46and EU position
 176. 40. The heterodimeric antibody of claim 26 furthercomprising a second heavy chain and a first light chain and a secondlight chain, wherein the second heavy chain comprises amino acidsubstitutions at AHo position 46 and EU positions 183, 392 and 409, andwherein the first and second light chains comprise an amino acidsubstitution at AHo position 46 and EU position 176, wherein the aminoacid substitutions introduce a charged amino acid at said positions. 41.The heterodimeric antibody of claim 40, wherein glutamine at positionAHo 46 of the first heavy chain is replaced with glutamic acid, whereinglutamine at position AHo 46 of the second heavy chain is replaced withlysine, wherein glutamine at position AHo 46 of the first light chain isreplaced with lysine, wherein glutamine at position AHo 46 of the secondlight chain is replaced with glutamic acid, wherein serine at EUposition 183 of the first heavy chain is replaced with glutamic acid,wherein glutamic acid at EU position 356 of the first heavy chain isreplaced with lysine, wherein glutamic acid at EU position 399 of thefirst heavy chain is replaced with lysine, wherein serine at EU position183 of the second heavy chain is replaced with lysine, wherein lysine atEU position 392 of the second heavy chain is replaced with asparticacid, and wherein lysine at EU position 409 of the second heavy chain isreplaced with aspartic acid.
 42. A heterodimeric antibody that bindssclerostin and DKK-1, comprising a first heavy chain comprising avariable region amino acid sequence selected from the group consistingof SEQ ID NOs: 378 and 366 and comprising amino acid substitutions at EUpositions 183, 356 and 399 of the first heavy chain, a second heavychain comprising a variable region amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1003 and 974 and comprising amino acidsubstitutions at EU positions 183, 392 and 409 of the second heavychain, a first light chain comprising a variable region amino acidsequence selected from the group consisting of SEQ ID NOs: 376 and 364and comprising an amino acid substitution at EU position 176 of thefirst light chain, and a second light chain comprising a variable regionamino acid sequence selected from the group consisting of SEQ ID NOs:1002 and 978 and comprising an amino acid substitution at EU position176 of the second light chain; wherein the amino acid substitutionsintroduce a charged amino acid at said positions.
 43. (canceled)
 44. Anantibody that binds to a region of sclerostin comprising amino acids86-111 of SEQ ID NO: 1, wherein the antibody comprises a heavy chainhaving a CH3 domain comprising one or more amino acid substitutions,wherein the one or more substitutions introduce charged amino acids thatare electrostatically unfavorable to homodimer formation andelectrostatically favorable to heterodimer formation.
 45. The antibodyof claim 44, wherein a negative charged amino acid in the CH3 domain issubstituted with a positive charged amino acid.
 46. The antibody ofclaim 45, wherein the negative charged amino acid is at EU positionD399, E356, or E357.
 47. (canceled)
 48. The antibody of claim 45,wherein the positive charged amino acid is lysine.
 49. The antibody ofclaim 44, wherein a positive charge amino acid in the CH3 domain issubstituted with a negative charged amino acid.
 50. The antibody ofclaim 49, wherein the positive charged amino acid is at EU positionK370, K392 or K409.
 51. (canceled)
 52. The antibody of claim 49, whereinthe negative charged amino acid is aspartic acid.
 53. The heterodimericantibody of claim 40, wherein the heterodimeric antibody binds to aregion of sclerostin comprising amino acids 86-111 of SEQ ID NO: 1,wherein the first heavy chain and the first light chain comprise asclerostin-binding portion and the second heavy chain and the secondlight chain comprise a DKK1-binding portion.
 54. A heterodimericantibody that binds to a region of sclerostin comprising amino acids86-111 of SEQ ID NO: 1, the antibody comprising a first heavy chain anda first light chain comprising a sclerostin-binding portion and a secondheavy chain and a second light chain comprising a DKK1-binding portion,wherein the first heavy chain comprises amino acid substitutions at EUpositions 183, 356 and 399, wherein the second heavy chain comprisesamino acid substitutions at EU positions 183, 392 and 409, and whereinthe first and second light chains comprise an amino acid substitution atEU position 176, wherein the amino acid substitutions introduce acharged amino acid at said positions.
 55. The heterodimeric antibody ofclaim 1, wherein the antibody binds to a region of sclerostin comprisingamino acids 86-111 of SEQ ID NO:
 1. 56. The heterodimeric antibody ofclaim 1, that is an IgG immunoglobulin.
 57. A nucleic acid comprising anucleotide sequence encoding the heterodimeric antibody according toclaim
 1. 58. A vector comprising the nucleotide sequence of claim 57.59. An isolated host cell comprising the nucleic acid of claim
 57. 60. Amethod of increasing bone mineral density in a mammalian subjectcomprising administering the heterodimeric antibody of claim 1 to thesubject in an amount effective to increase bone mineral density in thesubject.
 61. A composition comprising the antibody of claim 1 and apharmaceutically acceptable carrier, diluent or adjuvant.
 62. Thecomposition according to claim 61, wherein less than 5% of the antibodyin the composition is in aggregate form after two weeks of storage atabout 4° C.
 63. (canceled)
 64. The composition according to claim 62,wherein an amount of antibody in the composition present in aggregateform in determined by Size Exclusion Chromatography.
 65. (canceled) 66.(canceled)
 67. (canceled)